the molecular mechanisms underlying skeletal and cardiac
TRANSCRIPT
i
The molecular mechanisms underlying skeletal and cardiac muscle
remodeling in the hibernating thirteen-lined ground squirrel
By
Yichi (Tony) Zhang
B.Sc. Kinesiology, Queen’s University, 2014
A Thesis Submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the
requirements for the degree of
Master of Science
Department of Biology
Carleton University
Ottawa, Ontario, Canada
© 2016
Yichi Zhang
ii
The undersigned hereby recommend to the Faculty of Graduate Studies and Research acceptance
of his thesis
The molecular mechanisms underlying skeletal and cardiac muscle
remodeling in the hibernating thirteen-lined ground squirrel
Submitted by
Yichi (Tony) Zhang
In partial fulfillment of the requirements for the degree of Master of Science
_________________________________
Chair, Department of Biology
_________________________________
Thesis Supervisor
Carleton University
iii
Abstract
The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) survives winters by hibernating,
whereby body temperature (Tb) cycles between 4ºC during torpor and 37ºC during arousal. Each
organ/tissue of the hibernator must make specific adjustments that allow the ground squirrel to
maintain or readjust physiological function during hibernation. The remodeling that occurs in
skeletal and cardiac muscle is unique to hibernators, and it is fascinating as a natural means of
avoiding physiological dysfunction in these tissues. The purpose of this thesis is to evaluate the
molecular mechanisms underlying muscle remodeling in both tissues. It was identified that
calcium signaling activates the NFAT-calcineurin pathway, leading to increased expression of
hypertrophy-promoting targets in both skeletal and cardiac muscle during torpor. In addition, we
found that there is differential expression and activity of transcription factors (Foxo, MyoG) and
ubiquitin ligases (MAFbx and MURF1) that promote muscle atrophy in the two tissues being
studied.
iv
Acknowledgements
First off, I would like to thank Dr. Ken Storey for graciously offering to take me on as a Master’s
student and for his constant support and mentorship. This next paragraph is dedicated to thanking
the man, the myth, the legend. Throughout the summer of 2014, I contemplated where I wanted
to start my Master’s, and to be honest I was initially very unsure of my decision to come to
Carleton. I knew that I love basic science, but I wanted to apply my findings to medicine, and so
I thought long and hard about starting a Master’s at the UOttawa Faculty of Medicine. However,
now that I look back on it, I couldn’t be happier with my decision to join the Storey Lab. Initially
it didn’t start off so great, I was preoccupied with rowing, volunteering, and shadowing doctors
while getting “my” work done at the lab. I know that you wanted me to be more of a leader, but
that wasn’t what I wanted from my Master’s – I wanted to fly under the radar, get my results and
publications, then get into medical school (I’m always going to be honest with you, no BS).
Despite this disagreement, you let me do what I do best, which is focusing on my own bench
work, writing it up and publishing it, then plan the subsequent projects and repeating this process
over and over again. I had a very systematic approach to my Master’s, and you let me focus on
my approach without much deviation (i.e. spending a lot of time teaching undergrads). This is
what makes you a great lab manager and teacher; you know the individual strengths of your team
members, and you let them play their role. While I decide what to do next, I just want to let you
know that my accomplishments and success over the past two years would not have been
possible had I not decided joined the Storey Lab under your tutelage. Therefore, should I be
blessed with an opportunity to take the next step in my pilgrimage (to become an orthopedic
surgeon and NBA team doctor one day), I’d say that half of the credit for me getting this far has
to go to you. I will continue to look up to you as a mentor, teacher, and role-model.
I will remember how you take every opportunity to chirp Lebron and my affection for him. To be
honest, I love LeBron the person more so than the basketball player, but I want and need to see
him succeed in bringing a title back to Cleveland because I want to know that a kind,
philanthropic, deeply friend-oriented, family man and good guy in general can make it to the top
of the mountain and be recognized as one of the greatest if not the greatest alongside all the cold-
blooded killers in the history books – Jordan, Curry, Bird, Kareem, as well as the ringless
Iverson and Westbrook – who are recognized for their greatness. Lastly, you’ve asked me a few
times to consider staying in the lab for a PhD, and the reason I can’t do it, as you probably
already know, is because I have not only big and ambitious plans outside of research, but within
research as well. I believe that the only way to get better and improve in your craft is to test
yourself, to challenge and learn from those who are better than you at what you do. Therefore,
should I continue down the research path and start a PhD, I need to test myself against the top
medical researchers in the business so that I know if I’m cut out for doing this for the rest of my
life. You are like Dirk Nowitzki, who is a legend, a revolutionary player, and someone who has
stayed true to himself and became the best at what he does (just like you have focused on the
same field of research and became the best in your field). But as you know, I identify more with
LeBron, and so I need to test myself against all the greats in pursuit of becoming the G.O.A.T
(Greatest of All Time). Call me conceded, but although I know this journey is going to be
stressful and difficult, my goals have kept me and will keep me going.
Ok, so after that long epiphany, I must also thank the others who have contributed to my
accomplishments and the fun (sometimes too much fun) times I have had over the past two
v
years. Thanks first of all to Jan Storey, mother of dragons, although most of us in the lab are just
simple hatchlings (I think there’s even a few dragons who are still in their shell…). Your edits
taught me what an excellent manuscript and poster should look like; you made me a better writer
and researcher. Also, you are a human encyclopedia and Ken knows (even if he doesn’t show it
sometimes) that you are the engine that keeps the lab running. However, Sanoji W is starting to
exert her dominance and take control of your dragons as their step-mother! Beware!
To my dear friends in the lab, all of you have helped me improve as a researcher and as a person
in one way or another. Some have provided me with constant support (even when I didn’t need
it), shout out to Sanoji W, Kama S, and Rasha A (the Ugly Sisters from Cinderella though you’re
anything but ugly). Some have changed my views and values for the better through our DMCs
(Deep Meaningful Conversations), holler at Mike S (aka BFF), Bryan L (aka Luu), and Alex W
(aka Abdul). Some have changed my views and values for the worse; don’t worry this isn’t a bad
thing Sam W; I’d rather see myself live long enough to become a villain than die a hero. Also
shout out to Sam L and Hanane HM for checking my privilege and commanding my respect with
your hard work and dedication #thefuture. There are plenty of people I haven’t gotten the chance
to thank, but that doesn’t mean I don’t appreciate your support. I’m the type of person who is
introverted and likes to keep things to himself. You might not see me as such, but trust me on
this one, I wish I got to know everyone in the lab equally but I can’t help but be the way I am.
Sanoji W knows this better than anyone, and despite your flaws (even though you think you’re
flawless), I know that your meddling in my business is your way of showing that you care. For
that, I will always cherish your friendship. Lastly, thank you to the undergrads in the Storey Lab
as well, you know who you are! Storey Lab will always be a kid’s zone, never change!
I would like to thank my rowing team for motivating me, teaching me, and supporting me.
You’re a fun group of cats, especially when we’re not waking up at 4 am to get to practice and
row at freezing temperatures. Thanks to those on the team who played intramural basketball in
our offseason and especially to Mikayla Arends for putting together the team, it was a blast (even
if we only won one game). Thanks to Coach Ed for putting up with all the times we broke our
boats and oars, and with all my missed practices when I had pneumonia. Thanks to Coach Matt
Noël for teaching this novice to row. Thanks to Coach Martin Rowland for teaching me how to
erg properly…somewhat (lol, bad habits are hard to correct). Special shout out to Michael
Mikolainis, Jeffrey Parkhouse, and Jason Sukstorf for making the transition from Novice to
Varsity with me. Also, shout out to Vince O’Shaughnessey for being my pair (that never actually
raced) partner. Lastly, Darren Major, you da best. Also, fyi Ken, we do not just row around
pointlessly in circles every morning, if anything we row in rectangles. Get your geometry right!
Lastly, I would like to thank my parents, without whom I would not have been blessed with life,
and none of this would have been possible. Thanks to my dad for being the glue that holds this
family together, and to my mom for always pushing me to work harder and to reach for the stars.
Thanks also to all my friends, especially those who rose up with me during our undergrad at
Queen’s, and my friends at UOttawa and Carleton as well. You are truly the ones who kept me
sane when I felt like I was going insane, you who gave me hope when I felt hopeless. Regardless
of whether or not we will be colleagues in the future, I will take every opportunity to repay you
for all the kindness and support you have shown me.
“Be so good they can’t ignore you”
- Damian Lillard aka Dame Dolla
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Table of Contents
Title Page
i
Abstract
iii
Acknowledgements
iv
Table of Contents
vi
List of Abbreviations
vii
List of Figures
xii
List of Appendices
Chapter 1
General Introduction 1
Chapter 2
General Materials and Methods 16
Chapter 3
Expression of nuclear factor of activated T cells
and downstream muscle-specific proteins in ground
squirrel skeletal muscle during hibernation
26
Chapter 4
Nuclear factor of activated T cells regulates cardiac
hypertrophy through calcium signaling during
hibernation
52
Chapter 5
Regulation of Foxo4 and MyoG promotes skeletal
muscle atrophy during torpor in ground squirrels
64
Chapter 6
Transcriptional activation of muscle atrophy
promotes cardiac muscle remodeling during
mammalian hibernation in ground squirrels
82
Chapter 7
General Discussion
100
Publication List
117
References
121
Appendices 145
vii
List of Abbreviations
AIH – autoinhibitory domain
AMPK - AMP-activated protein kinase
APS – ammonium persulfate
ATP – adenosine triphosphate
bp – base pairs
CAM - calmodulin
CAMKIV – calmodulin-dependent protein kinase IV
CBP – CREB-binding protein
CDK - cyclin-dependent kinase
ddH2O – double distilled water
DPI-ELISA – DNA-protein interaction enzyme-linked immunosorbent assay
DTT - dithiolthreitol
Dyrk1a – dual-specificity tyrosine-(Y)-phosphorylation regulated kinase
EA – early arousal
EC – euthermic control
EDTA – ethylenediamine tetraacetic acid
viii
EMBL-EBI – European Molecular Biology Laboratory - European Bioinformatics Institute
EN – entrance into torpor
ER – euthermic room temperature
ET – early torpor
Foxo - forkhead box transcription factors of the O subclass
GSK3β – glycogen synthase kinase 3 beta
GTP – guanosine triphosphate
H2O2 – hydrogen peroxide
HDAC – histone deacetylase
Hepes – 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HRP – horse radish peroxidase
HSF1 – heat shock transcription factor 1
JNK - Jun N-terminal kinase
IA – interbout arousal
iTRAQ – isobaric tag for relative and absolute quantification technology
kDa - kilodalton
LA – late arousal
ix
LT – late torpor
MAFbx - muscle atrophy F-Box
MEF2 - myocyte enhancer factor-2
MHC – myosin heavy chain
MK - MAPK-activated protein kinase
MLC – myosin light chain
MURF1 - muscle Ring Finger 1
MyoD – muscle differentiation protein
MyoG – myogenin
NIH – National Institute of Health
NINDS – National Institute of Neurological Disorders and Stroke
NCBI – National Center for Biotechnology Information
NFAT - nuclear factor of activated T cells
PAGE – polyacrylamide gel electrophoresis
PBS – phosphate buffer saline
PBST – phosphate buffer saline with Tween
PGC-1α – PPARγ coactivator 1-alpha
PI3K – Phosphoinositide 3-kinase
x
PKA – protein kinase A
PKB/Akt – protein kinase B
PMSF – phenylmethanesulfonylfluoride
PPARγ – peroxisome proliferator-activated receptor gamma
PVDF – polyvinylidine fluoride
Ralbp1 – Ral binding protein 1
RT – room temperature
SDS – sodium dodecyl sulfate/lauryl sulphate
Ser – serine
Tb – body temperature
TBS – tris buffer saline
TBST – tris buffer saline with Tween
TEMED – N, N, N’, N’-tetramethylethylenediamine
TF - transcription factors
Thr – threonine
TMB - tetramethylbenzidine
TNFα - tumor Necrosis Factor α
xi
Tris – tris (hydroxymethyl) aminomethane
USDA – United State Department of Agriculture
UPS – ubiquitin proteasome system
xii
List of Figures
Figure 1.1
Ground squirrel avoidance of skeletal muscle wasting.
11
Figure 1.2
Ground squirrel cardiac dimensions during active season and
hibernation.
12
Figure 1.3
Schematic representation of the NFAT-calcineurin pathway
and its regulation.
13
Figure 1.4
Signaling pathways involved in the regulation of muscle
atrophy through the ubiquitin proteasome system (UPS).
14
Figure 1.5
Model for Ras-Ral pathway-dependent Foxo4 regulation of
skeletal muscle atrophy.
15
Figure 2.1
Schematic depiction of the hibernation torpor-arousal cycle.
25
Figure 3.1
DNA sequence analysis of the myomaker promoter in multiple
animals to find putative NFAT binding sites in ground
squirrel.
45
Figure 3.2
Changes in NFAT protein levels in ground squirrel skeletal
muscle over the torpor-arousal cycle.
46
Figure 3.3
Changes in myoferlin and myomaker protein levels in ground
squirrel skeletal muscle over the torpor-arousal cycle.
47
Figure 3.4
Changes in calcineurin, CAM, and calpain1 protein levels in
ground squirrel skeletal muscle over the torpor-arousal cycle.
48
Figure 3.5
Changes in NFAT-binding to DNA in ground squirrel skeletal
muscle over the torpor-arousal cycle.
49
Figure 3.6
Effect of adjusting temperature on NFAT-DNA binding in
ground squirrel skeletal muscle over the torpor-arousal cycle.
50
Figure 3.7
Effect of adding urea and Ca2+ on NFAT-DNA binding in
ground squirrel skeletal muscle over the torpor-arousal cycle.
51
Figure 4.1
Changes in the protein levels of NFATs in ground squirrel
cardiac muscle over the torpor-arousal cycle.
61
Figure 4.2
Changes in myoferlin and myomaker protein levels in ground
squirrel cardiac muscle over the torpor-arousal cycle.
62
xiii
Figure 4.3
Changes in calcineurin, CAM, and calpain protein levels in
ground squirrel cardiac muscle over the torpor-arousal cycle.
63
Figure 5.1 Changes in the protein levels of Foxo4 and p-Foxo4 in ground
squirrel skeletal muscle over the torpor-arousal cycle.
78
Figure 5.2 Changes in phosphorylation ratios of Foxo4 in ground squirrel
skeletal muscle over the torpor-arousal cycle.
79
Figure 5.3 Changes in the protein levels of MyoG, MAFbx, and MURF1
in ground squirrel skeletal muscle over the torpor-arousal
cycle.
80
Figure 5.4 Changes in the protein levels of Ras, Ral, and Ralbp1 in
ground squirrel skeletal muscle over the torpor-arousal cycle.
81
Figure 6.1 Changes in the protein levels of Foxo1 and p-Foxo1 in ground
squirrel cardiac muscle over the torpor-arousal cycle.
95
Figure 6.2 Changes in the protein levels of Foxo3a and p-Foxo3a in
ground squirrel cardiac muscle over the torpor-arousal cycle.
96
Figure 6.3 Changes in phosphorylation ratios of Foxo1 and 3a in ground
squirrel cardiac muscle over the torpor-arousal cycle.
97
Figure 6.4 Changes in the protein levels of Foxo4 and MyoG in ground
squirrel cardiac muscle over the torpor-arousal cycle.
98
Figure 6.5 Changes in the protein levels of MAFbx and MURF1 in
ground squirrel cardiac muscle over the torpor-arousal cycle.
99
Figure S.1
Table S.1
Summary figure of the relationship between the targets
analyzed in the current thesis.
Table summarizing the changes that took place with western
blotting results of the NFAT-calcineurin pathway and their
downstream muscle-specific proteins in skeletal muscle.
147
148
Table S.2
Table S.3
Table summarizing the changes that took place with the DPI-
ELISA results of NFATc1, c3, and c4 in skeletal muscle.
Table summarizing the changes that took place with the
western blotting results of the NFAT-calcineurin pathway and
their downstream muscle-specific proteins in cardiac muscle.
149
150
xiv
Table S.4
Table S.5
Table summarizing the changes that took place with the
western blotting results of the Ras-Ral pathway, Foxo4 and its
phosphorylated forms, as well as Myogenin their downstream
E3 ubiquitin ligase proteins in skeletal muscle.
Table summarizing the changes that took place with the
western blotting results of Foxo1, Foxo3a, Foxo4 and its
phosphorylated forms, as well as Myogenin their downstream
E3 ubiquitin ligase proteins in cardiac muscle.
151
152
2
Physiological adaptations (i.e. hibernation, freezing, and estivation) to environmental
changes is vital to the survival of many if not all organisms. This is especially true for organisms
that face extreme environmental challenges, which have developed a range of adaptations to
ensure their survival. One such adaptation used by some mammals in order to survive prolonged
seasonal exposure to stressful environmental conditions such as lack of food, frigid temperatures,
and so on, is hibernation. The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) is an
excellent example of a hibernating mammal as these animals are native to the central prairies of
North America, and they survive winters by hibernating underground. In preparation for
hibernation, ground squirrels enter a state of hyperphagia where excessive eating results in large
increases in body weight by up to 40% (Storey, 2010). During hibernation, these animals
undergo cycles of torpor and arousal. During torpor, the animals suppress their metabolic rate
(often to just 2-4% of normal conditions) and drop their core Tb from 35-38˚C to levels that
match the ambient temperature of its surroundings (as low as 0-5˚C) (Frerichs & Hallenbeck,
1998; Storey & Storey, 2004; Storey, 2010; Wang & Lee, 2011). During the process of
metabolic rate depression within torpor, most physiological functions are reduced; respiration
rates (approximately 2.5% of euthermia), organ perfusion (<10% of euthermia), neuron firing,
and a shift in the metabolic profile from a reliance on carbohydrate metabolism to fat metabolism
via β-oxidation are such examples (Buck & Barnes, 2000; McArthur & Milsom, 1991; Storey &
Storey, 2004). Prolonged periods of torpor (often 1-2 weeks or more) are interspersed with brief
periods of arousal where metabolic rate and Tb return to euthermic levels.
This strategy for conserving energy can save hibernating ground squirrels up to 88% of
the ATP expenditure that would otherwise be required to maintain euthermic physiological
conditions over the winter months (Wang & Lee, 2011). Metabolic rate suppression is a
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controlled process that maintains cellular homeostasis at colder Tb while simultaneously
reprioritizing ATP use by different cell functions. These functions include cell preservation
strategies such as antioxidant defense, differential regulation of enzymes by mechanisms like
reversible protein phosphorylation, differential regulation of protein chaperones, in addition to
differential regulation of transcription factors that modulate the expression of selected genes
and/or proteins that support cell- or tissue-specific needs (Fahlman, Storey, & Storey, 2000;
MacDonald & Storey, 2005; Mamady & Storey, 2006; Morin & Storey, 2006; Morin, Ni,
McMullen, & Storey, 2008).
During hibernation, each organ/tissue of the hibernator must make specific adjustments
that allow them to maintain or preserve physiological function under the periods of low Tb
during torpor, the fluctuations in Tb as a result of torpor-arousal cycles, and various cellular
stresses.
Skeletal Muscle
The skeletal muscle experiences muscle wasting, whereby reductions in muscle mass,
strength, and the relative amount of slow oxidative fiber occur when a prolonged period of
mechanical unloading or inactivity occurs (Bassel-Duby & Olson, 2006; Choi, Selpides, Nowell,
& Rourke, 2009; Malatesta, Perdoni, Battistelli, Muller, & Zancanaro, 2009; Rourke, Yokoyama,
Milsom, & Caiozzo, 2004). For hibernators, this muscle wasting would be highly
disadvantageous, because following hibernation, these animals need to resume natural activities
and scavenge for food right away. Therefore, there is a need to reduce significant losses in
muscle mass during hibernation. Interestingly, these animals seem to be able to do just that as
numerous studies have demonstrated a lack of significant muscle wasting during hibernation
despite the prolonged periods of inactivity and mechanical unloading that occur during
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hibernation (Cotton & Harlow, 2015; Gao et al., 2012; Xu et al., 2013). As shown in Figure 1.1,
the relative ratio of muscle mass/body weight actually increases throughout hibernation in
ground squirrels due to significant losses in body weight as well as an extremely effective
mechanism of muscle preservation and remodeling that is unique to hibernators (Gao et al.,
2012). As one would expect, nitrogen balance appears to be maintained throughout hibernation
due to a balance between protein synthesis and breakdown in the skeletal muscle (Lee et al.,
2012). In summary, hibernators have unique mechanisms of muscle remodeling and preservation
in comparison with non-hibernating mammals such as humans. We set out to elucidate the
molecular mechanisms underlying this process with the hope that our findings could become
applicable to the identification of novel targets for the treatment of muscle wasting diseases such
as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy (Boyer et al., 2014; Haslett et
al., 2003).
Cardiac Muscle
Another organ that must make significant adjustments during hibernation is the heart.
During torpor, the squirrel’s heart rate is strongly reduced, often from euthermic rates of 350-400
beats/min to just 5-10 beats/min. These changes in heart rate, in addition to the increased
viscosity of blood at low Tb, require significant changes in cardiovascular dynamics (Frerichs &
Hallenbeck, 1998; Frerichs, Kennedy, Sokoloff, & Hallenbeck, 1994). The strength of each
individual contraction must be significantly greater as a result of pressure and volume overloads,
therefore cardiac hypertrophy is observed during torpor (Depre et al., 2006). In humans, cardiac
hypertrophy is often characterized by significant cardiac fibrosis whereby collagen deposition
occurs to stiffen cardiac chamber walls, reduce diastolic filling, and ultimately preventing the
heart from pumping enough blood to meet bodily demands. This is a condition known as heart
5
failure, and it is caused by abnormal metabolic, structural, and functional events occurring in the
heart (Day, 2013; Hill and Olson, 2008). What is fascinating about the hearts of ground squirrels
is that their cardiac dimensions (left ventricular mass, internal dimensions, and wall dimensions)
are increased during hibernation in comparison with active, non-hibernating squirrels (Nelson &
Rourke, 2013) (Figure 1.2). This phenomenon suggests that ground squirrels have an efficient
mechanism of cardiac remodeling, whereby it can undergo cardiac hypertrophy when necessary
to maintain perfusion and then reverse this process after hibernation. The molecular basis behind
this mechanism has yet to be discovered and understanding how this process occurs could
provide novel insight into the development and treatment of maladaptive cardiac hypertrophy
and heart failure.
Objectives and Hypotheses
Although metabolic rate depression in I. tridecemlineatus is characterized by a global
suppression of most processes that cause ATP expenditure, including important functions such as
transcription and translation, positive regulation of select genes and proteins still occur in order
to ensure the animal’s survival. As a matter of fact, various approaches of gene- and protein-
screening have identified various targets that are upregulated during hibernation (Li et al., 2013;
Storey & Storey, 2010). These targets include transcription factors (TFs) that play an integral
part in regulating gene transcription. Since these TFs regulate the transcription of specific genes
and groups of genes that play vital roles in the cell, I decided to focus on studying TFs that
regulate the expression of genes/proteins that play important roles in muscle remodeling.
Characterizing the expression and activity of these TFs and their downstream targets over the
torpor-arousal cycle will improve upon our current knowledge of the molecular mechanisms
underlying the unique physiological processes that occur in ground squirrel skeletal and cardiac
6
muscles. Given the lack of cures for diseases and conditions such as Spinal Muscular Atrophy,
Duchenne Muscular Dystrophy, and heart failure, there is a need to identify novel targets for
therapeutic intervention and to improve our understanding of the mechanisms underlying muscle
remodeling (Boyer et al., 2014; Day, 2013; Haslett et al., 2003). Therefore, these are the
challenges that motivate me to study and compare these two different types of muscle and how
the squirrel is able to naturally avoid physiological dysfunction in these tissues.
Regulation of the nuclear factor of activated T cells (NFAT)
Currently, there are many TFs that have been implicated in muscle remodeling and they
seem to play vital roles in both hypertrophy and atrophy, these TFs include MEF2, MyoG, Foxo,
and NFAT (Armand et al., 2008; Boyer et al., 2014; Day, 2013; Haslett et al., 2003). The NFATs
in particular are a family of transcription factors that have been implicated in multiple aspects of
skeletal muscle remodeling; including hypertrophy, fiber-type switching, and myogenesis
(Armand et al., 2008; Delling et al., 2000; Hudson et al., 2014). For instance, NFATc2-null mice
have defective myoblast fusion and myogenesis, resulting in fibers with reduced size and delayed
repair in response to injury (Horsley et al., 2001). In cardiomyocytes, NFATs regulate
cardiomyocyte atrophy, apoptosis, development, and growth (Li et al., 2013; Lin et al., 2009;
Liu, Wilkins, Lee, Ichijo, & Molkentin, 2006; Molkentin et al., 1998; Schubert et al., 2003). In
fact, in another hibernating animal – the woodchuck (Mormota monax) – Li et al. (2013) used
iTRAQ technology and mass spectrometry to identify that there is an upregulation in the NFAT
pathway during hibernation in the hearts of these animals. Inactive NFATs are located in the
cytoplasm and are heavily phosphorylated, and NFATs are activated and nuclear localization
occurs via dephosphorylation by calcineurin (Park et al., 2000), a calmodulin-stimulated protein
phosphatase, allowing NFATs to translocate to the nucleus and bind to transcriptional complexes
7
of their target genes (Rusnak & Mertz, 2000) (Figure 1.3). Therefore, the rationale behind why
we decided to study this family of TFs specifically with regard to muscle remodeling during
hibernation is due to its role as a master regulator of muscle remodeling and its identification as a
potential target through proteomics studies.
Hypothesis 1: I. tridecemlineatus undergoes significant skeletal muscle remodeling
(simultaneous muscle protein synthesis and breakdown) in order to avoid disuse-induced skeletal
muscle wasting during torpor. The NFAT-calcineurin pathway will be activated by calcium
signaling during torpor in order to upregulate muscle specific genes that play a role in muscle
hypertrophy and/or maintenance.
Chapter 3 explores this hypothesis by examining the protein levels (via immunoblotting)
of NFATs, their downstream muscle specific targets, and calcium signaling factors that are
upstream of NFATs in ground squirrel skeletal muscle over the torpor-arousal cycle.
Furthermore, nuclear localization of NFATs and their binding activities (defined as the ability of
transcription factors to bind to their conserved promoter sequence) were examined using DNA-
Protein Interaction (DPI) ELISA assays to get a sense of NFAT activity, which is more important
than NFAT levels with regard to their role in regulating gene transcription. We found that the
NFAT TFs showed increased binding activity that was initiated by increased levels of Ca2+-
signaling proteins upon entering torpor. There were modest increases in NFAT protein levels as
well during torpor, and as a result the expression of muscle specific proteins were highly
upregulated during torpor. In addition, we also found that the cellular environment has an effect
on NFAT-binding to DNA; specifically, temperature and calcium concentration both affect
NFAT activity.
8
Hypothesis 2: I. tridecemlineatus undergoes cardiac muscle remodeling in order to maintain
cardiovascular function during torpor through cardiac hypertrophy and to avoid chronic
maladaptive cardiac hypertrophy by reversing this process during arousal. The NFAT-
calcineurin pathway will be activated by calcium signaling during torpor in order to upregulate
muscle specific genes that play a role in cardiac hypertrophy, and this molecular response should
differ from those of skeletal muscle.
Chapter 4 explores this hypothesis by examining the protein levels (via immunoblotting)
of NFATs, their downstream targets in cardiac muscle, and calcium signaling factors that are
upstream of NFATs in ground squirrel skeletal muscle over the torpor-arousal cycle. There were
modest increases in NFAT protein levels as well during torpor and downregulation during
arousal, especially in NFATc2 and c3, which are the NFATs that are most important in
regulating the expression of genes important for muscle growth and development (Armand et al.,
2008; Delling et al., 2000; Lin et al., 2009). The decreases in NFATc2 and c3 protein levels
corresponded with a decline in the expression of calcium signaling factors, indicating that
calcium signaling regulation of NFATs may play a role in the reversal of cardiac hypertrophy.
Furthermore, the expression of muscle specific proteins were highly upregulated only during
early torpor, and the pattern of expression was different from that of skeletal muscle.
Regulation of forkhead box O (Foxo) and Myogenin (MyoG)
Other sets of transcription factors that play key roles in the regulation of muscle
remodeling include MyoG and the Foxo family of TFs (Moresi et al., 2010; Sandri et al., 2004;
Stitt et al., 2004). Recent studies have begun to elucidate the molecular basis of both skeletal and
cardiac muscle remodeling in hibernators, with findings indicating that the peroxisome
proliferator-activated receptor γ coactivator 1-α (PGC-1α) and NFATs are implicated in this
9
process (Li et al., 2013; Xu et al., 2013; Zhang & Storey, 2015). However, a current gap in
knowledge exists around whether the molecular pathways of muscle atrophy and protein
degradation are activated in skeletal and cardiac muscle during hibernation as a result of
inactivity and cardiac hypertrophy. The main signaling pathway that controls muscle atrophy
involves the Foxo as well as the MyoG TFs, and their regulation of the ubiquitin proteasome
system (UPS) (Moresi et al., 2010; Sandri et al., 2004; Stitt et al., 2004). The UPS is an
important mechanism for protein degradation, whereby substrates are ligated to ubiquitin via E3
ubiquitin ligases like MAFbx/atrogin-1 and MURF1, which target these substrates for
degradation in the proteasome. These ligases have been studied extensively in relation to muscle
atrophy as they have been shown to degrade muscle proteins like MHC as well as MLC-1 and -2
(Foletta, White, Larsen, Léger, & Russell, 2011; Herrmann, Lerman, & Lerman, 2007;
Schiaffino, Dyar, Ciciliot, Blaauw, & Sandri, 2013). Due to the importance of both MAFbx and
MURF1 for muscle atrophy, common regulators were found for both ligases. The Foxo family of
TFs were the first of such factors (Moresi et al., 2010; Sandri et al., 2004; Stitt et al., 2004).
Then, MyoG, which was initially identified as a regulator of myogenesis, was shown to be a
positive regulator of both E3 ligases as well; where the expression of MAFbx and MURF1, as
well as muscle atrophy were attenuated in MyoG-null mice (Moresi et al., 2010). The
mammalian Foxo family has four members: Foxo1, Foxo3a, Foxo4, and Foxo6, that are involved
in various cellular processes in addition to muscle atrophy, such as antioxidant defense and
apoptosis (Birkenkamp & Coffer, 2003; Greer & Brunet, 2005; Wu & Storey, 2014). Foxo1,
Foxo3a, and Foxo4 are all regulated by the Akt/protein kinase B (PKB) signaling pathway,
which is activated by PI3K in the presence of insulin (Burgering and Eijkelenboom, 2013).
Specifically, Akt blocks the function of all three Foxo proteins through phosphorylation at
10
conserved residues that lead to cytoplasmic localization (Brunet et al., 1999; Matsuzaki, Ichino,
Hayashi, Yamamoto, & Kikkawa, 2005; Takaishi et al., 1999; Tang, Nuñez, Barr, & Guan,
1999) (Figure 1.4). However, Foxo4 transcriptional activity has been shown to be regulated
through a separate pathway as well, involving the Ras and Ral GTPases, as well (De Ruiter,
Burgering, & Bos, 2001; Essers et al., 2004; Kops et al., 1999; Van Den Berg et al., 2013)
(Figure 1.5).
Hypothesis 3: It is hypothesized that during the avoidance of muscle loss during hibernation,
muscle atrophy signaling pathways regulating the UPS will either be inhibited or be stable during
torpor. It is expected that differential regulation of MAFbx and MURF1 expression through
Foxo4 and MyoG will be observed, and that Foxo4 dephosphorylation through inhibition of the
Ras-Ral pathway will play a role in this process.
Chapter 5 explores this hypothesis by examining the protein levels (via immunoblotting)
of Foxo4, phosphorylated Foxo4 (p-Foxo4), MyoG, MAFbx, MURF1, as well as the main
factors involved in the Ras-Ral pathway (Ras, Ral, Ralbp1), in ground squirrel skeletal muscle
over the torpor-arousal cycle. Furthermore, nuclear localization of Foxo4 and its activity with
regard to its ability to regulating gene transcription will be characterized by assessing the levels
of phosphorylated Foxo4 at different residues. We identified that although there is an increase in
Foxo4 protein levels during torpor, there is a greater decrease in activated Foxo4 (p-Foxo4
T451). Therefore, there is a decrease in Foxo4 activity during torpor, which is reflected by
decreases in Ras, Ral, and Ralbp1 protein levels during torpor as well. This decrease in Foxo4
activity, coupled with decreases in MyoG protein levels during torpor, resulted in decreased
expression in both MAFbx and MURF1 during torpor, especially at early torpor (ET).
11
Hypothesis 4: It is predicted that I. tridecemlineatus avoids reverses cardiac hypertrophy by
activating muscle atrophy signaling pathways as they are aroused from torpor. The Foxo family
of transcription factors as well as MyoG are believed to play a significant role in this reversal of
cardiac hypertrophy through their regulation of the UPS.
Chapter 6 explores this hypothesis by examining the protein levels of the Foxo family of
transcription factors (Foxo1, 3a, 4) as well as their phosphorylated forms via immunoblotting. In
addition, levels of MyoG and the ubiquitin ligases MAFbx and MURF were characterized as
well. Foxo1 and 3a nuclear translocation was assessed by characterizing the p-Foxo levels as
Akt/PKB phosphorylates Foxo1 and 3a at the residues tested in order to prevent nuclear
translocation (Dobson et al., 2011). Immunoblotting results demonstrated that there is
upregulation of Foxo1 and 3a protein levels as well as decreases in inactive, phosphorylated
Foxo1 and 3a proteins during torpor in comparison with euthermic control. Foxo4 and MyoG on
the other hand increased in late torpor. MAFbx and MURF1 showed a similar pattern of
expression where their protein levels increased in late torpor as well as arousal, thus suggesting
that the ubiquitin proteasome system (UPS) is activated as ground squirrel are aroused from
torpor, which could be causing muscle atrophy and the reversal of cardiac hypertrophy.
Figures
12
Figure 1.1: Ground squirrel body mass, muscle wet mass, and muscle-to-body weight ratio
before, during, and after hibernation (mean ± SD, N = 8 each). EDL – Extensor Digitorum
Longus. Adapted from (Gao et al., 2012).
Figure 1.2: Left ventricular internal diameter dimension (LV i.d.), LV wall dimensions (LVW),
LV mass, and left atrium (LA) to aortic (Ao) root dimension are greater in hibernation than
control in squirrels. *p<0.05. Adapted from (Nelson & Rourke, 2013).
13
Figure 1.3: Schematic diagram of the calcineurin-NFAT pathway and its regulation by Ca2+
signaling in myocytes (muscle cells). Ca2+ uptake by myocytes activates calmodulin and
calpain1, which activate calcineurin as a result. Activated calcineurin removes the phosphate
group on NFAT transcription factors, allowing for nuclear translocation, where it can regulate
the expression of genes essential for muscle hypertrophy. When intracellular Ca2+ levels
decrease and calcineurin becomes inactive, and NFAT is phosphorylated and exported by several
different kinases including GSK3β, PKA, and Dyrk1a.
14
Figure 1.4: Signaling pathways involved in E3 ubiquitin ligase (MAFbx and MURF1) regulation
which result in physiological outcomes such as muscle remodeling and protein
degradation/atrophy in skeletal and heart muscle. Transcriptional regulation of E3 ligases occur
via several transcription factors (TFs) such as Foxos and myogenin. Several kinases have the
ability to inhibit Foxo activation and translocation to the nucleus through phosphorylation on
several residues for Foxo1, 3a, and 4.
15
Figure 1.5: Model for Akt-dependent and Ras-Ral pathway-dependent FOXO4 regulation of
muscle atrophy. Cellular stresses (i.e. hyperoxia, increase in Tb) that induce production of
reactive oxygen species (ROS) results in activation of the Ras-Ral pathway. Activated RalA then
regulates the assembly, activation, and phosphorylation of JNK onto the JIP1 scaffold, possibly
involving Ralbp1, a protein commonly found in association with RalA. Phosphorylated JNK is
able to phosphorylate and activate Foxo4 at Threonine 451 so that it can regulate the expression
of E3 ubiquitin ligases like MAFbx/atrogin-1 and MURF1, thus promoting muscle atrophy.
Phosphorylation of Foxo4 at Serine 197 by Akt results in cytoplasmic localization, thus
preventing Foxo4 from translocating to the nucleus and regulating transcription.
17
Animals
Thirteen-lined ground squirrels (I. tridecemlineatus), which weighed 150-300 g, were
wild-captured by the United States Department of Agriculture (USDA) licensed trappers (TLS
Research, Bloomingdale, IL). Animals were then transported to the Animal Hibernation Facility
at the National Institute of Neurological Disorders and Stroke (NINDS, Bethesda, MD), where
all experiments were conducted by the laboratory of Dr. J.M. Hallenbeck as previously described
(McMullen & Hallenbeck, 2010). All animal procedures were approved by the Animal Care and
Use Committee of the National Institute of Neurological Disorders and Stroke (NIH; animal
protocol no. ASP 1223-05). Male and female ground squirrels were sampled equally in the study
with a mixture of genders in each experimental condition and all animals were between 1-3 years
of age, although the exact age of the animals is unknown since animals were wild-captured. At
NINDS, animals were housed individually in cages in a holding room with a constant ambient
temperature of 21ºC under a 12h light: 12h dark cycle. Animals were fitted with a sterile
programmable temperature transponder (IPTT-300; Bio Medic Data Systems) injected
subcutaneously in the intrascapular area while the squirrels were anaesthetized with 5%
isofluorane. Animals were fed water and standard rodent chow ad libitum until they gained
sufficient lipid stores to enter hibernation.
To enable a natural transition into torpor, animals were transferred to constant darkness in
an environmental chamber at 4-5°C at the end of October. To not disturb the torpid squirrels, a
red safe light (3-5 lux) was used when entering the chamber and a heavy dark curtain was used to
shield the shelves containing the cages and block the light and sound resulting from opening and
closing the door to the environmental chamber. Body temperature (Tb), time elapsed, and
18
respiration rates were monitored and used to determine the stage of torpor-arousal cycle. All
animals had been through torpor-arousal bouts prior to sampling, therefore they were deep in
hibernation when sampling took place. Four different animals were euthanized and tissue
samples were collected at the following sampling points: 1) Euthermic Room temperature (ER);
these animals were held in the holding room with an ambient temperature of 21°C (Tb=~37°C).
Tissues were collected from animals at this time point after they had reached their plateau
weight. 2) EC designates euthermic in the cold room. These squirrels had a stable Tb of 37°C for
at least three days and were capable of entering torpor, but had not re-entered hibernation in the
past 72 h. These euthermic animals displayed slow-wave sleep characteristics that were observed
in all sampling animals, and thus were chosen as the reference group to eliminate compounding
variables of environmental light, temperature, feeding, in addition to time/season. 3) EN
designates entrance into hibernation; entrance into the torpor-arousal cycle is characterized by
falling Tb with sampling occurring between 31° and 18°C. 4) ET designates early torpor;
squirrels had entered torpor with a stable Tb at 5-8°C for ~24 h. 5) LT designates late torpor;
animals maintained a Tb at 5-8°C for >5 days. 6) EA designates early arousal; animals with a Tb
rising to at least ~12°C with increasing respiration to at least 60 breaths/min after torpor, 7) LA
designates late arousal; animals with increased respiration rate and Tb of 28-32°C. 8) IA
designates interbout arousal; animals were naturally aroused after the torpor phase of the
hibernation bout and reached the respiratory rate, metabolic rate, and body temperature of fully
aroused animals for 6 hours after being in torpor for at least 5 days. These animals remain in the
hibernaculum (4°C) but their core body temperature is back to ~37°C. The skeletal muscle was
collected and used for analysis from a mixture of hind limb muscles whereas the cardiac muscle
19
was collected from a mixture of atrial and ventricular tissue. A diagram of these torpor-arousal
cycle stages is shown in Figure 2.1, which was adapted from Tessier & Storey (2016).
Total Protein Extract Preparation
Total soluble protein extracts were prepared as previously described (Zhang & Storey,
2015) for samples of frozen skeletal muscle and heart from 4 animals for each stage in the
torpor-arousal cycle, where samples were collected from EC, EN, ET, LT, and EA for both
tissues. LA and ER were collected only for skeletal muscle and IA was collected only for cardiac
muscle due to limited tissue samples. Frozen samples of ~0.5g tissue were quickly weighed,
powdered into small pieces under liquid nitrogen and then homogenized (using a Polytron
PT10)1:3 w:v in ice-cold homogenizing buffer (20 mM Hepes, 200 mM NaCl, 0.1 mM EDTA,
10 mM NaF, 1 mM Na3VO4, 10 mM β-glycerophosphate at a pH of 7.5) with 1 mM
phenylmethylsulfonyl fluoride (Bioshop) and 1 μL/mL protease inhibitor cocktail (Bioshop)
added. Samples were centrifuged at 10,000 rpm for 10 min at 4°C and supernatants were
removed. Soluble protein concentration was assayed using the BioRad reagent (BioRad
Laboratories, Hercules, CA; Cat #500-0006) at 595 nm on a MR5000 microplate reader. Samples
were then adjusted to a final protein concentration of 10 μg/μL by the addition of a small volume
of homogenizing buffer and then aliquots were combined 1:1 v:v with 2x SDS loading buffer
(100 mM Tris-base, pH 6.8, 4% w:v SDS, 20% v:v glycerol, 0.2% w:v bromophenol blue, 10%
v:v 2-mercaptoethanol) and then boiled. The final protein samples at a concentration of 5 μg/μL
were stored at -20°C until use.
20
Preparation of Nuclear Protein Extracts
Nuclear protein extracts were prepared as previously described (Zhang & Storey, 2015)
and were separately extracted from the skeletal muscle of 4 animals for each of the seven
experimental stages (ER, EC, EN, ET, LT, EA, LA). Frozen skeletal muscle samples were
homogenized 1:2 w:v using a non-mechanical Dounce homogenizer (5 piston strokes) in lysis
buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 10 mM EDTA, 20 mM β-glycerophosphate), with
10 μL of 100mM DTT, 10 μL of protease inhibitor cocktail added immediately before
homogenization. Samples were centrifuged for 10 min at 10,000 rpm and 4°C and supernatants
were removed as the cytoplasmic fraction. Pellets were resuspended in 147 μL of nuclear
extraction buffer (20mM HEPES, pH 7.9, 400mM NaCl, 1 mM EDTA, 10% v/v glycerol, 20mM
β-glycerophosphate) with 1.5 μL of 100mM DTT, and 1.5 μL of protease inhibitor cocktail
added. Samples were incubated on ice with gentle rocking for 1 h and then centrifuged for 10
min at 10,000 rpm at 4°C. Protein concentrations were determined with the Bio-Rad protein
assay, adjusted to 5 μg/μL, and samples were stored at -80°C until use.
Western Blotting
The BioRad Mini Protean III system was used for SDS-PAGE. Equal amounts of protein
from each sample (25 -35 μg depending on the protein tested) were loaded onto 6-
15%polyacrylamide gels (depending on the protein tested) and were run at 180 V for 60-180
min. Polyacrylamide gels were made based on a discontinuous gel system, which included the
stacking gel at pH 6.8 (130 μl 1.0 M Tris-HCl, 170 μl 30 % acrylamide, 680 μl water, 10 μl 10%
SDS, 10 μl 10% APS, 1 μl TEMED) and a resolving gel at pH 8.8 (1.3 ml 1.5 M Tris-base, 1.7
ml 30% acrylamide, 2.0 ml water, 50 μl 10% SDS, 50 μl 10% APS, 2 μl TEMED, this is the
21
recipe for a 10% gel). The running buffer was diluted 10-fold from the stock solution (25.5 g
Tris-base, 460 g glycine, 25 g SDS, adjusted to 2.5 L with water) before use. Proteins were then
transferred to PVDF membranes by electroblotting at 160 mA for 60-180 min depending on the
protein tested or at 30 V for 100 min for small molecular weight proteins using a transfer buffer
containing 25 mM Tris (pH 8.5), 192 mM glycine and 10% v:v methanol at room temperature.
Membranes were then blocked for 30 min with 2.5-10% w:v milk, depending on the protein
tested, in 1x TBST (20 mM Tris base, pH 7.6, 140 mM NaCl, 0.05% v:v Tween-20, 90% v:v
ddH2O). After washing for 3 x 5 min again with 1x TBST, membranes were probed with specific
primary antibodies at 4°C overnight at a concentration of 1:500-1:1000 depending on the protein
tested. After probing with primary antibody, membranes were washed for 3 x 5 min with 1x
TBST and then incubated with HRP-linked anti-rabbit or anti-goat IgG secondary antibody
(Bioshop: 1:6000 v:v dilution) for 30 min at room temperature. After a second set of three
washes, bands were visualized by enhanced chemiluminescence (H2O2 and Luminol). Then,
blots were stained using Coomassie blue (0.25% w/v Coomassie brilliant blue, 7.5% v/v acetic
acid, 50% methanol) to visualize total protein levels. Immunoblot bands for ground squirrel
proteins corresponded to the molecular weights indicated on the respective antibody
specification sheets or the amino acid sequence of the I. tridecemlineatus isoform, as confirmed
by running PINK Plus Prestained Protein Ladder (FroggaBio) or BLUeye Prestained Protein
Ladder (FroggaBio) for high molecular weight proteins.
DNA-Protein Interaction (DPI)-ELISA
DNA oligonucleotides were designed based on the DNA binding elements of NFATc1-4
and were produced by Sigma Genosys (Oakville, ON, Canada). The biotinylated probe (NFAT
22
5’-Biotin-GGGAAGGAAAGTGCGGGTGG-3’) and the complement probe (NFAT 5’-Biotin
CCACCCGCACCCTTTTTCCC-3’) were first diluted in sterile water (500 pmol/μl), and the two
probes were mixed 1:1 v:v for a total of 20 μl. Probes were then placed in a thermocycler for 10
min at 94°C and gradually cooled to room temperature. Double stranded DNA probes were
diluted in 1x PBS (137 mM NaCl, 2.7 mM KCL, 10 mM Na2HPO4, pH 7.4), and 50 μl of diluted
DNA probe was added (40 pmol DNA/well) to streptavidin-coated wells on a microplate.
Following a 1 h incubation, unbound probe was discarded and wells were rinsed twice with 1x
wash buffer (1X PBS containing 0.1% Tween-20), and a third time with 1X PBS. Transcription
factor binding buffer (10 mM HEPES, 50 mM KCL, 0.5 mM EDTA, 3 mM MgCl2, 10% v/v
glycerol, 0.5 mg/ml bovine serum albumin, 0.05% NP-40, 0.5 mM DTT, 20 pg/μl Salmon Sperm
DNA, 44mM NaCl, pH 7.9) was added to each well containing the DNA probe along with 27.5
μg of the nuclear protein extract. Two negative control wells were loaded with transcription
factor binding buffer but no protein. Following another 1 h incubation with gentle shaking,
protein mixtures were discarded and the wells were washed three times with 1x wash buffer.
Diluted primary antibody (1:500) was then added (60 μl/well) for 1 h and were then
discarded, and wells were rinsed three times with 1x wash buffer before incubation with diluted
secondary (1:1000, 60 μl/well) for 1 h. This antibody was then discarded and wells were rinsed
three times with wash buffer. After secondary antibody incubation and washing, bound antibody
was detected using tetramethylbenzidine (TMB) (Bioshop). A 60 μl aliquot of TMB was added
to each well, colour was developed for 10-15 min, and then the reaction was stopped with 60 μl
of 1 M HCl. Absorbance was measured at 450 nm (reference wavelength of 655 nm) using a
Multiskan spectrophotometer. To control for background absorbance and non-specific binding,
test strip ELISA experiments were run with negative controls containing no probe or no protein
23
or no primary antibody added being run in duplicates using a pooled sample of multiple
sampling points. Conditions were optimized such that negative control wells showed >50%
decreases in absorbance relative to sample wells before quantification runs of sampling points
were conducted.
Environmental DPI-ELISA
To assess how TF-DNA binding is altered when environmental conditions (temperature,
[CA2+], [urea]) are altered, the DPI-ELISA protocol described above was modified. To test for
the effect of temperature on TF-DNA binding, the initial DNA probe synthesis, incubation, and
washing steps were carried out as previously described. Afterwards, TF binding buffer was
added to each well containing the DNA probe, plus one other well that is used to monitor
solution temperature. Buffer temperature was monitored using a digital thermometer with two
probes, one placed outside the solution to monitor ambient temperature and the other placed
inside the well to monitor solution temperature. The ELISA plate was placed in either a 4°C
fridge, a 37°C incubator, or left at room temperature. When the solution temperature has
matched and stabilized to the ambient temperature inside the fridge or incubator, 27.5 μg of
nuclear extracts of EC and LT samples were added to wells containing the DNA probe with the
exception of the duplicate negative controls, and the plates were placed on shakers. Following
the one hour incubation, all plates were placed at room temperature and the rest of the procedure
was performed as described above. Temperature DPI-ELISAs were performed to evaluate: 1) the
effect of temperature (37, 21, 4ºC) on TF-DNA binding for EC samples, 2) the effect of
temperature (37, 21, 4ºC) on binding for LT samples, and 3) the difference in binding between
EC and LT sampling points using their physiological temperatures, 37 and 4ºC, respectively.
24
In order to test for the effect of Ca2+ or urea on TF-DNA binding, the DPI-ELISA
protocol described above was followed; adjusting the TF binding buffer by adding Ca2+ or urea.
To assess the effect of Ca2+ on TF-DNA binding, quantification runs were performed on four
biological replicates of the LT samples with no protein and no Ca2+ (negative controls), no Ca2+,
100nM Ca2+, and 600nM Ca2+ added to the TF binding buffer during the protein incubation step.
100nM and 600nM of Ca2+ were selected because they represent the minimum and maximum
concentrations of nuclear Ca2+ that have been identified mathematically and experimentally
(Brière, Xiong, Mazars, & Ranjeva, 2006; Choi, Swanson, & Gilroy, 2011; Dobi & Agoston,
1998; Xiong, Tao, DePinho, & Dong, 2012). A similar experiment was performed to assess for
the effect of urea on TF-DNA binding, with quantification runs being conducted for LT samples
(n=4) with no protein and no urea (negative controls), no urea, 5mM urea, and 100mM urea
added to the transcription factor binding buffer during the protein incubation step. These two
concentrations were tested as 5mM is approximately the normal physiological concentrations of
serum urea in hibernating mammals (Chilian & Tollefson, 1976; Kristofferson, 1963; Stenvinkel
et al., 2013). Also, 100mM was shown experimentally as the maximum concentration of urea
that could be supplemented to media before cell culture growth and survival was inhibited
(Yancey & Burg, 1990).
Quantification and Statistics
Band densities on chemiluminescent immunoblots were visualized using a Chemi-Genius
BioImaging system (Syngene, Frederick, MD) and quantified using the Gene Tools software.
Immunoblot band density in each lane was standardized against the summed intensity of a group
of Coomassie-stained protein bands in the same lane; this group of bands was chosen because
25
they were not located close to the protein band of interest but were prominent and constant
across all samples. This method of standardizing against a total protein loading control has been
suggested to be more accurate in comparison with standardizing against housekeeping proteins
such as tubulin (Eaton et al., 2013). For DPI-ELISA experiments, quantification runs,
absorbance readings were corrected by subtracting optical density (OD) values for each sampling
point from OD values of blank wells containing no protein, and these values were then
normalized relative to EC. Similarly, western blot band densities were also normalized at each
other sampling point relative to EC. Immunoblotting and absorbance data are expressed as means
± SEM, n=4 independent samples from different animals. Statistical testing used the one-way
ANOVA and the Tukey post-hoc functions from the GraphPad Prism software (San Diego, CA).
Figures
Figure 2.1: Schematic depiction of the hibernation bout indicating time points and body
temperatures when animals were sacrificed. EC: euthermic in the cold room; EN: entrance into
Torpor; ET: early Torpor; LT: late Torpor; EA: early Arousal; LA: late Arousal; IA: interbout
arousal. Not shown, ER: euthermic at room temperature; this time point occurs before EC pre-
hibernation and after IA post-hibernation. Total time between ET and LT is at least 5 days.
Figure from Tessier & Storey (2016).
26
Chapter 3
Expression of nuclear factor of activated T cells and downstream
muscle-specific proteins in ground squirrel skeletal muscle during
hibernation
27
Introduction
The present chapter investigates how the skeletal muscle of thirteen-lined ground
squirrels adapts on a molecular level to maintain function at low Tb to support long-term torpor.
As mentioned in chapter 1, one potential issue affecting skeletal muscle during hibernation is
disuse-induced muscle wasting, which occurs commonly after long periods of inactivity and
results in reduced muscle mass, strength, and relative amount of slow oxidative muscle (Bassel-
Duby & Olson, 2006; Choi et al., 2009; Malatesta et al., 2009; Rourke et al., 2004). What makes
hibernating mammals, specifically the thirteen-lined ground squirrel, interesting as a model to
study muscle biology is that they demonstrate a lack of skeletal muscle atrophy despite
prolonged periods of mechanical unloading that occur over long periods of hibernation (Cotton
& Harlow, 2015; Gao et al., 2012; Xu et al., 2013) (Figure 1.1).
The present chapter focuses on the roles and regulation of the nuclear factor of activated
T cells (NFAT) family of transcription factors in I. tridecemlineatus, as they have been
implicated as a key regulator of skeletal muscle hypertrophy (Armand et al., 2008; Delling et al.,
2000; Hudson et al., 2014; Schiaffino, Sandri, & Murgia, 2007; Zhang & Storey, 2015). The
NFAT family contains five members named NFAT1-5 or NFATc1-4 and NFAT5, with
NFATc1-4 being regulated primarily by calcineurin (Rao, Luo, & Hogan, 1997). Calcineurin is a
CAM-stimulated protein phosphatase that regulates NFAT through dephosphorylation, thereby
activating and allowing NFAT to translocate to the nucleus and regulate gene transcription
(Rusnak & Mertz, 2000). CAM is a ubiquitously expressed Ca2+-binding protein that is involved
in a variety of signaling pathways that are Ca2+-dependent. It contains four EF-hand motifs, each
of which binds a Ca2+ ion (Kretsinger, 1987). CAM regulates calcineurin by binding to the
regulatory domain of the calcineurin A subunit when it is exposed due to conformational changes
28
caused by activation of the calcineurin B subunit when there is an increase in intracellular Ca2+
levels (Klee, Crouch, & Krinks, 1979; Yang & Klee, 2000). When calcineurin B binds to Ca2+
ions, a conformational change occurs in its C-terminal autoinhibitory domain, and the Ca2+-
dependent cysteine protease calpain, specifically calpain1/calpain-µ, cleaves the autoinhibitory
domain, thus activating calcineurin (Burkard, 2005; Lee et al., 2014; Shioda, Moriguchi,
Shirasaki, & Fukunaga, 2006). Therefore, both calmodulin and calpain1 are important regulators
of the NFAT-calcineurin pathway (Figure 1.3).
We sought to identify the role of Ca2+ signaling factors such as calmodulin, calpain1, and
calcineurin on NFAT transcriptional regulation of muscle-specific proteins in the skeletal muscle
of thirteen-lined ground squirrels. One such protein is myoferlin, a protein that is highly
expressed in skeletal muscle and to a lesser degree in cardiac muscle (Davis, Delmonte, Ly, &
McNally, 2000). However, myoferlin is highly expressed specifically in myoblasts undergoing
fusion, where it localizes at the sites of apposed membranes undergoing fusion (Davis, Doherty,
Delmonte, & McNally, 2002; Doherty et al., 2005). Furthermore, myoferlin mRNA was up-
regulated in human muscle affected by Duchenne muscular dystrophy (Haslett et al., 2003).
Myoferlin contains multiple NFAT-binding sites in its promoter, which drove high levels of
myoferlin expression in vitro and in vivo. Furthermore, expression was elevated in response to
muscle damage (Demonbreun et al., 2010). In myoferlin-null mice, muscle fiber size was
reduced due to impaired myoblast fusion, but the mice were still viable. However, myomaker, a
membrane protein found in the muscle that also controls myoblast fusion results in the complete
loss of myoblast fusion when it is mutated, resulting in the absence of all skeletal muscle, which
leads to postnatal death in myomaker-null mice (Millay et al., 2013). In addition, myomaker
expression and increased myoblast fusion has been shown to occur in adult satellite cells
29
following muscle injury (Millay, Sutherland, Bassel-Duby, & Olson, 2014). The regulation of
myomaker is not well-understood due to its recent discovery, with only two MRFs, muscle
differentiation protein (MyoD) and myogenin (MyoG), being known to induce myomaker
transcription (Millay et al., 2014). However, NFAT has been shown to regulate the expression of
MyoG cooperatively with MyoD (Armand et al., 2008).
In addition to affecting myomaker expression through NFATc2 regulation of MyoG, we
also used a DNA-protein interaction (DPI) enzyme-linked immunosorbent assay (ELISA) to test
the ability of NFATs to bind to a novel putative NFAT-binding sequence in the myomaker
promoter. Since activation of the NFAT-calcineurin pathway ultimately leads to increased
binding of NFAT transcription factors to its target promoters, we also used this technique to
analyze transcription factor binding activity to DNA because of its simplicity (Brand et al., 2013;
Brand, Kirchler, Hummel, Chaban, & Wanke, 2010; Jagelska, Brázda, Pospisilová, Vojtesek, &
Palecek, 2002). Given the extreme environmental stressors confronting 13-lined ground squirrels
during hibernation, we suspected that environmental factors such as temperature could
potentially affect the binding ability of NFATs and potentially other transcription factors, to
DNA. Recent literature has begun to show that gene expression could be affected by
temperature, but no study has directly investigated the temperature dependence of transcription
factor-binding to DNA (Novák et al., 2015; Chen, Nolte, & Schlötterer, 2015; Riehle, Bennett,
Lenski, & Long, 2003; Swindell, Huebner, & Weber, 2007). Most of these studies use DNA
microarrays to study the global changes in gene expression when temperature stress is induced
on an organism (Riehle et al., 2003; Swindell et al., 2007). However, although this approach
identifies targets that may be involved in stress-response, it does not directly elucidate
mechanisms such as transcription factor binding affinity. In addition to the ground squirrel’s
30
ability to thermoregulate during torpor-arousal cycles, they also show enhanced capabilities to
maintaining intracellular Ca2+ and urea concentrations in comparison with non-hibernating
animals under the same temperature stress (Chilian & Tollefson, 1976; Kristofferson, 1963; Liu,
Wang, & Belke, 1991; Wang & Zhou, 1999; Wang, Zhou, & Qian, 1999; Wang, Lakatta, Cheng,
& Zhou, 2002). Therefore, we adapted our DPI-ELISA protocol in order to run these
environmental ELISAs that allow us to characterize the effects of temperature and different
cellular metabolites such as Ca2+, and urea on transcription factor-DNA binding.
To explore the role of calcium signaling in activating the NFAT-calcineurin pathway and
their downstream muscle-specific targets, we quantified relative protein levels via
immunoblotting and utilized the DPI-ELISA technique to measure changes in TF-binding to an
oligonucleotide containing the NFAT response element. We predicted that the NFAT-calcineurin
pathway would be activated through upregulation of Ca2+ signaling proteins during torpor, and
that this would cause an upregulation of the muscle proteins, myoferlin and myomaker. The
secondary objective of this study was to identify the impact of environmental conditions on
NFAT-binding to target genes. We tested this theory using a modified, environmental DPI-
ELISA that allowed us to adjust the temperature and concentration of metabolites within the
assay.
Materials and Methods
Animals Experimental Conditions
All animals were captured, treated and their tissues were harvested following the same
protocol as previously described in Chapter 2. The skeletal muscle used was a mixture of several
hind limb muscles.
31
Total Protein and Nuclear Protein Extract Preparations
Total soluble protein extracts and nuclear protein extracts were prepared from frozen hind
leg skeletal muscle (approximately 500 mg) as previously described. Total protein extracts were
used for western blotting and nuclear protein extracts were used for DPI-ELISA experiments as
previously described in Chapter 2.
Western Blotting
Western blotting was performed as described in Chapter 2 for seven time points (ER, EC,
EN, ET, LT, EA, LA) for NFATc1-4, myoferlin, and myomaker and for six time points (EC, EN,
ET, LT, EA, LA) for calcineurin, CAM, and calpain1. Equal amounts of protein from each
sample (25 μg) were loaded onto 6% (NFATc1-4, myoferlin), 8% (calcineurin, calpain1), or 15%
(myomaker, CAM) polyacrylamide gels and were run at 180 V for 60-120 min. For calmodulin,
35 µg of protein for each sample were loaded on the polyacrylamide gels. Proteins were then
transferred to PVDF membranes by electroblotting at 320 mA for 60 min (myomaker), 90 min
(calcineurin, calpain1), 120 min (NFATc1-3, myoferlin), or at 30 V for 100 min (calmodulin).
Membranes incubated in primary antibodies were blocked for 30 min with 2.5% (for NFATc1-4)
or 5% (for CnA, CAM, calpain1, myoferlin and myomaker) w:v milk in 1x TBST.
Antibodies specific for mammalian NFAT-c1 (sc-13033), c2 (sc-13024), c3 (sc-8321), c4
(sc-13036), myoferlin (sc-134798), and myomaker (also known as TMEM8c, sc-244460) were
purchased from Santa Cruz Biotechnologies. All antibodies were used at a 1:500 v:v dilution in
1x TBST. A calmodulin (06-396) antibody from Upstate Biotechnology (Lake Placid, NY), as
well as calcineurin A (GTX111039) and calpain1 (GTX102340) antibodies from Genetex
(Irving, CA) were purchased and used at a 1:1000 v:v dilution in 1x TBST. All primary antibody
32
incubations took place over one night. Membranes that had been probed with myomaker
(TMEM8c) were incubated with HRP-linked anti-goat IgG secondary antibody (BioShop:
1:6000 v:v dilution). All other antibodies were detected using HRP-linked anti-rabbit IgG
secondary antibody (Bioshop: 1:6000 v:v dilution). All secondary antibody incubations were 30
minutes.
The primary antibodies cross-reacted with a single band on immunoblots at the expected
molecular masses from the antibody specification sheets. Other parts of the procedure were
carried out in accordance with the protocol described in Chapter 2.
DPI-ELISA and Environmental DPI-ELISA
DPI-ELISAs were performed as described in Chapter 2 for seven time points (ER, EC,
EN, ET, LT, EA, LA) for NFATc1-4. Environmental ELISAs were performed for NFATc1, c3,
and c4 using both the EC and LT time points to test for changes due to temperature (37, 21, 4ºC),
and changes due to changes in [Ca2] and [urea] were tested using the LT time point. Probes for
NFATc1-4 were purchased from Sigma Genosys including both the biotinylated probe (NFAT
5’-Biotin-GGGAAGGAAAGTGCGGGTGG-3’) and the complement probe (NFAT 5’-Biotin
CCACCCGCACCCTTTTTCCC-3’). These probes were designed using comparative sequence
analysis that identified conserved NFAT-binding sequences within the promoter of the
myomaker gene. Specifically, the total nucleotide sequences of human (Accession number:
NM_001080483.2), mouse (Accession number: NM_025376.3), and 13-lined ground squirrel
(Accession number: XM_005336956.1) myomaker were accessed from the NCBI Nucleotide
database and the sequence within 1500 base pairs (bp) upstream of the transcriptional start site
was downloaded. These upstream sequences were compared using the Vista program
33
(http://genome.lbl.gov/vista) with a window size of 100 bp and a minimum sequence identity of
70%. NFAT transcription factor binding sites were identified using the rVISTA program.
Predictions were made based on the TRANSFAC Professional 9.3 library using the default core
similarity value of 0.75 and the matrix similarity value of 0.70 as previously described
(Demonbreun et al., 2010).
Using rVista, all NFAT-DNA binding sites were identified and pairwise alignments were
conducted between myomaker upstream sequences in squirrel and mouse (Figure 3.1A), and in
squirrel and human (Figure 3.1B). The program displayed red lines identifying potential NFAT
binding sites that were aligned, allowing a maximum core shift of 6 bp and only one gap of any
length inside it. Squirrel-mouse and squirrel-human alignments had two NFAT binding sites in
common at 1095 and ~1440-bp upstream of the myomaker transcriptional start site, shown on
Figure 3.1 by the red lines. These two aligned NFAT binding sites were cross-referenced using
the conserved NFAT binding sequence from the literature (GGAAA) (Hung, Wang, Chang, &
Shyu, 2008; Rao et al., 1997). Only the 1095-bp upstream sequence remained a probable NFAT-
myomaker binding site (Figure 3.1C). Subsequently, a DNA oligonucleotide was designed for
this putative binding site (NFAT 5’-Biotin-GGGAAGGAAAGTGCGGGTGG-3’) containing the
general NFAT binding sequence and the flanking region specific to the myomaker promoter.
Findings indicated that NFATc1, c3, and c4 were able to bind specifically to the DNA
oligonucleotide, as NFATc2 did not pass the test strip stage, with negative control wells showing
high absorbance readings despite persistent optimization. The NFAT timecourse ELISA
quantification runs as well as environmental ELISA quantification runs for NFATc1, c3, and c4
utilized the following conditions; 40 pmol DNA/well, 27.5 µg of protein/well, 1 µg of salmon
sperm/well, 44 mM NaCl, 1:1000 v/v NFATc1, c3, and c4 primary antibody (same as those used
34
for western blotting) in 1x PBST, 1:1000 secondary antibody (same as those used for western
blotting) in 1x PBST. The rest of the protocol was followed as described in Chapter 2.
Quantification and Statistics
Band densities for NFATc1-4, myoferlin, myomaker, calcineurin, CAM, and calpain-1
on chemiluminescent immunoblots were visualized and quantified as described in Chapter 2.
Relative binding affinity for the DPI-ELISA was quantified as described in Chapter 2. Data are
expressed as means ± SEM, n=4 independent samples.
Results
Analysis of NFATc1-4 protein levels in skeletal muscle
NFATc1 protein levels decreased during torpor, reaching its lowest levels at LT (78%
lower in comparison with EC, p<0.05), before rising back up to EC levels during arousal.
NFATc2 levels on the other hand increased during torpor, peaking at LT (1.75 fold higher in
comparison with EN, p<0.05). NFATc3 levels were highest initially at ER (1.82 fold higher in
comparison with EC, p<0.05) before decreasing dramatically. Protein levels then decreased again
during LA (55% lower in comparison with LT). Finally, NFATc4 levels were highest at ER, then
levels rose from EC throughout the torpor-arousal cycle, peaking at LA (higher than EN by 1.97
fold, p<0.05) (Figure 3.2).
Analysis of myoferlin and myomaker (TMEM8c) protein levels
Myoferlin levels increased dramatically during torpor (EN, ET, LT increased by 3.45,
4.75, and 4.46 fold, respectively in comparison with EC, p<0.05) and then decreased upon
35
entering arousal. Myomaker on the other hand showed constant protein levels from throughout
the torpor-arousal cycle (Figure 3.3).
Calcineurin, Calmodulin, and Calpain Protein Levels
Calcineurin protein levels increased upon entering torpor (EN) by 1.19-fold in
comparison with euthermic control (EC), p<0.05. Protein levels then returned to baseline levels
at EC during early torpor (ET), but it spiked once more by 2.08-fold (in comparison with EC,
p<0.05) and reached its highest level during late torpor (LT). Upon entering arousal, calcineurin
levels decreased once again to baseline at early arousal (EA) and then increased once more by
1.2-fold (compared to EC, p<0.05) at late arousal (LA). Calpain1 showed a similar pattern of
expression, where spikes in protein levels occurred at EN, LT, and LA. Calpain levels increased
modestly at EN by 0.72-fold relative to EC. At LT, there was a greater increase of 2.37-fold
relative to EC (p<0.05). The final spike at LA was even greater, where protein levels increased
by 4.4-fold in comparison with EC (p<0.05). Calmodulin protein levels remained fairly constant
throughout the torpor-arousal cycle with the exception of EN, where levels increased by 2-fold
relative to EC (p<0.05) (Figure 3.4).
Analysis of NFATc1-4 Relative Binding to DNA
Relative binding to DNA (transcription factor activity) was measured for seven time
points: ER, EC, EN, ET, LT, EA, and LA. NFATc1 activity decreased by 58% and 54% at EN
and EA, respectively, relative to ER (p<0.05), but there was a progressive increase throughout
torpor from EN to LT. These decreases in DNA binding at EN and EA were seen in NFATc3 as
well (46% and 30% lower respectively relative to EC). In addition, there was a large increase in
NFATc3-DNA binding at LT (3.99-increase relative to EN, p<0.05). NFATc4 binding levels
36
decreased modestly by 63% at EN from EC, then binding increased dramatically by 3.96-fold
relative at ET relative to EN (p<0.05). Following ET, binding activity decreased slightly at LT,
then it increased slowly during EA and LA, with binding at LA being 3.77-fold greater than EN
(p<0.05).
Effect of Temperature on NFATc1, c3, and c4 Relative-Binding to DNA
Due to the drastic changes in Tb when ground squirrels enter torpor, we modified the
DPI-ELISA in order to study the effect of temperature on NFATc1, c3, and c4 transcription
factor binding to DNA. Three temperatures (37, room temperature – 21, and 4ºC) were studied at
the EC and LT sampling points. As mentioned previously, EC animals had not entered
hibernation yet, so their Tb remained at 37ºC. LT animals were at the deepest part of torpor;
where Tb was 4-5ºC (McMullen & Hallenbeck, 2010).
The temperature DPI-ELISA experiments performed on the EC time point showed that
NFATc1 and NFATc4 binding to DNA decreased dramatically (p<0.05) by 77% and 94%,
respectively, from 37ºC to room temperature. NFATc3 on the other hand, showed a modest
decrease of 37% in binding activity. However, when we compared changes in binding for all
three NFATs between 37 and 4ºC, they all showed significant decreases in binding (p<0.05) by
at least 84% (Figure 3.6A). Therefore, all three NFATs showed progressive declines in binding
activity at the EC time point as temperature was decreased. The temperature DPI-ELISA using
the LT sampling point showed a similar pattern for NFATc1 and c4, where binding decreased by
66% and 95%, respectively, from 37ºC to room temperature (Figure 3.6B). NFATc1 binding
levels decreased further from 37ºC to 4ºC by 86% (p<0.05). On the other hand, NFATc4 binding
levels stabilized from room temperature to 4ºC. For the LT sampling point, NFATc3 binding
levels did not seem to be affected much by the changes in temperature, as there was only a
37
modest decline in binding by 46% when comparing 37 to 4ºC (Figure 3.6B). While analyzing the
difference in transcription factor binding to DNA at physiological conditions from EC at 37ºC to
LT at 4ºC, we observed sharp declines in binding for both NFATc1 and c4 by 89% and 93%,
respectively (p<0.05). The decline in binding from EC to LT for NFATc3 was 64%, which is
less compared to the differences observed for NFATc1 and c4, but this difference was still
significant (p<0.05) (Figure 3.6C).
Effect of Ca2+ and Urea on NFATc1, c3, and c4 Relative-Binding to DNA
We tested for the effect of adding Ca2+ and urea to the DPI-ELISA assay in an attempt to
discover how these two metabolites/substrates effect NFATc1, c3, and c4 TF-DNA binding.
Urea and Ca2+ are of particular interest due to the unique changes in the animal’s regulation of
the urea cycle and Ca2+ signaling, which occur during mammalian hibernation (Chilian &
Tollefson, 1976; Epperson et al., 2011; Lee, Buck, Barnes, & O’Brien, 2012; Stenvinkel et al.,
2013; Wang et al., 1999; Wang et al., 2002). The addition of 5mM and 100mM of urea seemed
to have no effect on the binding of NFATc1, c3, or c4 to DNA, with binding levels remaining
stable throughout the different conditions during torpor (Figure 3.7A). When 100nM of Ca2+ was
added to the protein incubation, the binding of NFATc1 to DNA showed a sizeable difference
(42% decrease relative to the no Ca2+ control) out of the three NFATs tested at LT. When 600nM
of Ca2+ was added however, NFATc1-DNA binding continued to decrease (57% decrease
relative to control, p<0.05) with NFATc4 binding showing a 0.42-fold increase relative to the no
Ca2+ control. Throughout the conditions, NFATc3 binding to DNA did not change appreciably
during torpor (Figure 3.7B).
Discussion
38
The present study aimed at furthering our understanding of the molecular mechanisms
underlying muscle remodeling in both skeletal and cardiac muscle during hibernation in the 13-
lined ground squirrel. The ground squirrel provides an excellent natural model system for
studying muscle remodeling as the skeletal muscle appears to undergo alternating patterns of
atrophy and hypertrophy without a net loss in muscle mass and the cardiac muscle is known to
undergo reversible cardiac hypertrophy (Hindle et al., 2014; Li et al., 2013; Nelson & Rourke,
2013; Wickler, Hoyt, & van Breukelen, 1991). However, the molecular mechanisms that are
responsible the preservation and/or change of muscle structure/function during hibernation are
not well known. Therefore, the present study focuses on the family of transcription factors
known as NFATs, which have been shown to regulate targets associated with skeletal muscle
hypertrophy, apoptosis, and development (Armand et al., 2008; Delling et al., 2000; Hudson et
al., 2014; Li et al., 2013; Lin et al., 2009; Liu et al., 2006; Molkentin et al., 1998; Schiaffino et
al., 2007; Schubert et al., 2003; Tessier & Storey, 2012, 2010). Furthermore, the downstream
targets myoferlin and myomaker were evaluated because of their newly identified and crucial
role as muscle membrane proteins in muscle development and repair (Demonbreun et al., 2010;
Doherty et al., 2005; Millay et al., 2013, 2014).
The relationship between myoferlin and NFATc1-4 was established when it was
observed that the myoferlin promoter contains multiple NFAT-binding sites that were sufficient
to drive high levels of myoferlin expression, especially following muscle damage (Demonbreun
et al., 2010). However, the link between myomaker and NFAT remains more of a mystery.
Currently, only MyoD and MyoG have been shown to induce myomaker transcription due to its
very recent discovery (Millay et al., 2014). However, MyoD is a common co-factor of NFAT as
they can bind to the same transcriptional complex, and they have been shown to synergistically
39
regulate the transcription of myog (Armand et al., 2008). Comparative sequence analysis of the
myomaker promoter region for potential NFAT transcription factor binding domains resulted in
the identification of multiple sites with the consensus NFAT-binding sequence (GGAAA) from
literature (Hung et al., 2008; Rao et al., 1997). Following further diagnostic analysis of potential
NFAT-binding sites using the rVista alignment tool, one NFAT-binding domain 1095-bp
upstream of the myomaker transcriptional start site was identified as it contained the consensus
sequence in addition to showing conservation in squirrel-mouse and squirrel-human pairwise
alignments (Figure 3.1). Therefore, comparative sequence analysis of the myomaker promoter
region has resulted in the identification of a possible novel NFAT-myomaker binding site. Using
DNA-Protein binding (DPI)-ELISAs, NFATc1, c3, and c4 were then shown to bind to this site
differentially over the torpor-arousal cycle (Figure 3.5). DPI-ELISAs represent a simple and
efficient way to measure transcription factor binding activity by quantitatively measuring the
ability of transcription factors to bind to DNA from nuclear extracts. This is the first time that
NFAT binding to the myomaker promoter has ever been identified, and this novel finding could
lead to further studies that characterize NFAT regulation of this vital myogenic protein.
NFATs are regulated by calcineurin, a calmodulin-stimulated phosphatase that
dephosphorylates NFATs, thus allowing them to translocate into the nucleus (Rusnak & Mertz,
2000). Calcineurin is sensitive to changes in calcium levels and it is positively regulated by other
calcium-signalling proteins such as calmodulin (CaM) and calpain (Al-Shanti & Stewart, 2009;
Burkard, 2005; Lee et al., 2014; Shibasaki, Hallin, & Uchino, 2002; Shioda et al., 2006; Wu et
al., 2004; Yang & Klee, 2000). Therefore, experiments were conducted to study the role of
calcineurin, CAM, and calpain as upstream regulators of the NFAT pathway within the context
of muscle remodeling and maintenance. Due to the improved Ca2+-handling abilities of ground
40
squirrels in comparison with non-hibernating mammals, [Ca2+] changes very little from
euthermia to the 0-5°C Tb seen during torpor (Frerichs & Hallenbeck, 1998; Liu et al., 1991;
Wang & Lee, 2011; Wang & Zhou, 1999; Wang et al., 1999; Wang et al., 2002). However, the
amplitude of Ca2+ transients following excitation is actually increased following excitation at low
temperatures, and as a result, stronger contractions with higher amplitudes are seen at lower
temperatures (Liu, Wang, & Belke, 1993; Liu, Wohlfart, & Johansson, 1990; Wang, Zhou, &
Qian, 2000; Wang, Huang, Liu, & Zhou, 1997). Therefore, greater spikes in intracellular [Ca2+]
following an action potential leads to an activation of NFAT-calcineurin pathway, allowing for a
maintenance of muscle mass during hibernation.
Our results show that indeed, there is an upregulation of Ca2+ signaling proteins like
calmodulin and calpain1 during torpor, where protein levels increased by 2-fold and 0.72-fold
relative to EC, respectively, at EN. These increases are accompanied by a 1.19-fold rise
(compared to EC) in calcineurin levels downstream (Figure 3.4). As a result of this increase in
calcineurin levels and activity, NFATc1, c3, and c4 translocate to the nucleus and show increases
in binding to DNA during torpor (Figure 3.5). A similar pattern is seen during LA, where
calpain1 levels increased by 4.4-fold relative to EC and there was an accompanying increase in
calcineurin levels as well (1.2-fold compared to EC) (Figure 3.4). Once more, there was an
increase in NFATc1, c3, and c4 binding activity accompanying the upregulation and activation
of calcineurin at LA (Figure 3.5). This upregulation of the NFAT-calcineurin pathway during LA
is somewhat unexpected and could reflect the role that NFAT TFs play in not only muscle
remodeling, but the generation of reactive oxygen species, which are produced rapidly due to
oxidative thermogenesis in squirrels during arousal (Kalivendi et al., 2005).
41
Immunoblotting analysis showed elevations of NFATc2 at late torpor in the skeletal
muscle of ground squirrels. Although all NFATs are expressed in skeletal muscle and are
important for proper muscle function, NFATc2 and c3 are mostly commonly associated with
myoblast fusion and muscle repair (Armand et al., 2008; Cho et al., 2007; Demonbreun et al.,
2010; Horsley et al., 2001). Therefore, the increase in NFATc2 protein levels at ET (Figure 3.2)
and the increase in NFATc3-DNA binding at LT (Figure 3.5) are indicative of increased skeletal
muscle regeneration and repair in an effort to maintain skeletal muscle mass despite disuse-
induced muscle wasting during torpor (Hindle et al., 2014). During LT, 2.08-fold and 2.37-fold
rises in calcineurin and calpain1 levels respective, relative to EC were observed and this
correlates with the rise in NFATc3 (Figure 3.4). Similar to NFATc3-DNA binding, myoferlin
protein levels increased significantly during torpor as well; rising at EN, and peaking at ET and
LT before decreasing to euthermic levels (Figure 3.4). Therefore, despite not showing significant
differences in protein levels, the large increase in NFATc3 activity during torpor may be driving
the increase in myoferlin levels that maintain skeletal muscle mass (Demonbreun et al., 2010).
This increase in myoferlin supports the hypothesis that there is an increase in muscle
regeneration to preserve skeletal muscle during torpor.
Having established the important role of Ca2+ in regulating the NFAT-calcineurin
pathway and muscle maintenance during hibernation through Ca2+-binding proteins, we became
interested in determining whether Ca2+ can directly affect NFAT binding to target promoters
during torpor. Several studies have previously shown that intranuclear Ca2+ can regulate gene
expression by directly binding to DNA or through regulation of transcription factors and their co-
factors (Chawla, Hardingham, Quinn, & Bading, 1998; Dobi & Agoston, 1998; Pusl et al., 2002;
Thompson et al., 2003). We identified using a modified environmental DPI-ELISA that Ca2+ did
42
indeed affect the binding of NFAT transcription factors to DNA during torpor. It was observed
that progressively increasing [Ca2+] decreased the binding of NFATc1, whereas NFATc4 showed
increased binding to DNA when [Ca2+] was increased to 600nM (Figure 3.7B). These effects of
intranuclear Ca2+ on NFAT-DNA binding during torpor seem to be specific for each NFAT
transcription factor, therefore this effect is likely not due to the binding and blocking of DNA by
intranuclear Ca2+ (Dobi & Agoston, 1998). This effect is most likely due to Ca2+ regulation of
specific export kinases like calmodulin-dependent protein kinase IV (CAMKIV) or through
specific coactivators of individual NFATs, such as CREB-binding protein (CBP) (Chawla et al.,
1998; Yang, Davis, & Chow, 2001). Given that urea is another key metabolite that is crucial
during hibernation, specifically torpor, we created an environmental DPI-ELISA to test whether
urea could affect NFAT binding activity as well (Epperson et al., 2011; Stenvinkel et al., 2013).
Not surprisingly, urea did not have a significant effect on NFAT-binding to DNA in any of the
tested conditions during LT (Figure 3.7A). Theoretically, the nuclear membrane allows
compounds of 60 kDa or less to pass through into the nucleus, and urea is just over that cut-off
(Gerace & Burke, 1988). Therefore, it would not be able to translocate into the nucleus.
Given the extreme variations in temperature that occurs during torpor-arousal cycles from
euthermia (37ºC) to torpor (0-4ºC), we were interested in knowing whether temperature could
potentiate or inhibit the binding of transcription factors, such as NFATs (Frerichs & Hallenbeck,
1998; Storey & Storey, 2004; Storey, 2010; Wang & Lee, 2011). We carried out a modified DPI-
ELISA, adjusting for the ambient temperature during the protein incubation step where binding
between transcription factors and the DNA oligonucleotide occurs. We found that there were
dramatic differences in transcription factor binding of NFATc1, c3, and c4 as the temperature
was progressively decreased from euthermic (EC) Tb (37ºC) to the depressed Tb seen during LT
43
(4ºC) (Figure 3.6A-C). When comparing the declines in NFAT-DNA binding from EC to LT, we
can see that there was a lesser decrease in the binding activity of NFATc3 (64%) relative to
NFATc1 (89%) and NFATc4 (93%) (Figure 3.6C). This relatively smaller decrease in NFATc3
binding activity could partly explain the preservation of skeletal muscle mass during torpor, as
NFATc3 plays the most vital role in coordinating muscle remodeling out of the four NFATs
(Armand et al., 2008; Delling et al., 2000; Demonbreun et al., 2010; Hudson et al., 2014). Due to
metabolic rate depression and the need to conserve ATP during torpor, the expression of
nonessential genes is likely halted, therefore NFATc1 and c4 activity show a greater decline
compared to NFATc3. This study is the first to identify changes in transcription factor-DNA
binding affinity that are temperature-dependent, although further studies need to be conducted to
determine whether our findings are specific for NFAT transcription factors or if it reflects a
greater number of transcription factors. More importantly, further studies need to determine
whether the temperature-sensitivity of NFAT transcription factors are due to conformational
changes that occur at lower temperatures to the protein itself, to DNA, or if it has to do with
interactions with temperature-sensitive cofactors. For example, NFATc2 has been shown to
cooperate with heat shock transcription factor 1 (HSF1), which is responsible for regulating the
gene expression of other heat shock proteins (Hayashida et al., 2010).
In summary, the present study provides insight into some of the key proteins involved in
skeletal and cardiac muscle remodeling during hibernation. Although more remains to be
investigated, including the regulation of factors associated with the UPS (Chapter 5), the results
from this study indicate that NFATc1-4 as well as the targets myoferlin and myomaker are
important to the hypertrophy and preservation of skeletal muscle mass and function during
hibernation. Our findings also demonstrate that Ca2+ signaling plays a key role in regulating the
44
NFAT-calcineurin pathway in skeletal muscle over the torpor-arousal cycle. In addition, in this
chapter, a novel technique, the environmental DPI-ELISA, was developed and used to study the
effects of environmental stimuli such as temperature, [urea], and [Ca2+] on transcription factor
binding to target promoters, which was the secondary objective of this study. We found that
[urea] has little effect on NFAT binding, but intranuclear [Ca2+] seems to affect the DNA-
binding activity of both NFATs c1 and c4, possibly through Ca2+ regulation of export kinases
and coactivators. Furthermore, we determined that temperature differences from euthermia
(37ºC) to torpor (4ºC) have profound effects on the binding of NFATc1, c3, and c4 although the
effects are more pronounced for NFATs c1 and c4. The novel finding that TF-binding to DNA is
temperature dependent should be explored further for other TFs and to identify potential
mechanisms. These findings contribute to our understanding of muscle remodeling, and these
mechanisms involved in preserving ground squirrel skeletal muscle throughout the torpor-arousal
cycle make studying the ground squirrel biologically-relevant. Promising therapeutics for
muscle wasting include anabolic androgens, such as nandrolone, which has been shown to
activate calcineurin-NFAT signaling and reduced denervation-induced muscle atrophy in mice
(Qin, Pan, Wu, Bauman, & Cardozo, 2015).
45
Figures
Figure 3.1: DNA sequence analysis of the myomaker promoter in multiple animals to find
putative NFAT binding sites. The sequence 1500 bp upstream of the (a) squirrel and mouse, and
(b) squirrel and human myomaker transcriptional start sites were interrogated using rVista for the
detection of NFATc1-4 binding sequences. Red lines indicate aligned hits of possible NFATc1-4
binding regions between organisms, the two sequences highlighted were identified by rVista as
possible binding sites. Cross-referencing with NFAT binding sequence from literature (c, shown
in green), one probable binding site remained 1095 bp upstream of the myomaker transcriptional
start site, this sequences along with its aligned flanking region from the multiple alignments was
used to design a 25 bp probe for DNA ELISA. Figure from (Zhang & Storey, 2015).
46
Figure 3.2: Changes in the protein levels of NFAT transcription factors over the course of the
torpor-arousal cycle in skeletal muscle of I. tridecemlineatus. NFATc1, c2, c3, and c4 total
protein expression levels were visualized at seven sampling points: ER, EC, EN, ET, LT, EA,
LA. See Materials and methods for more extensive definitions. Representative Western blots and
Coomassie total protein loading controls are shown for selected pairs of sampling points that are
labeled to the left and right of the gel. Sample numbers (lanes) are labeled along the top
indicating 4 samples of one type (e.g., EC lanes 1, 2, 3, 4) and 4 samples of another (e.g. LA
lanes 5, 6, 7, 8). Also shown are histograms with mean standardized band densities (± S.E.M.,
n=4 independent protein isolations from different animals). Data was analyzed using a one-way
analysis of variance with a post hoc Tukey’s test (p<0.05); for each parameter measured, values
that are not statistically different from each other share the same letter notation. Figure from
(Zhang & Storey, 2015).
47
Figure 3.3: Changes in myoferlin and myomaker total protein levels in skeletal muscle over the
torpor-arousal cycle in I. tridecemlineatus. Myoferlin and Myomaker total protein expression
levels were visualized at seven sampling points: ER, EC, EN, ET, LT, EA, LA. Representative
Western blots and Coomassie total protein loading controls are shown for selected pairs of
sampling points that are labeled to the left and right of the gel. Sample numbers (lanes) are
labeled along the top indicating 4 samples of one type (e.g., EC lanes 1, 2, 3, 4) and 4 samples of
another (e.g. EN lanes 5, 6, 7, 8) using Myoferlin as an example. Also shown are histograms
with mean standardized band densities (± S.E.M., n=4 independent protein isolations from
different animals). Data was analyzed using a one-way analysis of variance with a post hoc
Tukey’s test (p<0.05); for each parameter measured, values that are not statistically different
from each other share the same letter notation. Figure from (Zhang & Storey, 2015).
48
Figure 3.4: Changes in calcineurin, calmodulin, and calpain1 total protein levels in skeletal
muscle over the torpor-arousal cycle in I. tridecemlineatus. Calcineurin, calmodulin, and
calpain1 total protein expression levels were visualized at six sampling points: EC, EN, ET, LT,
EA, LA. Representative Western blots and Coomassie total protein loading controls are shown
for selected pairs of sampling points that are labeled to the left and right of the gel. Sample
numbers (lanes) are labeled along the top indicating 4 samples of one type (e.g., EN lanes 1, 2, 3,
4) and 4 samples of another (e.g. ET lanes 5, 6, 7, 8) using Calcineurin as an example. Also
shown are histograms with mean standardized band densities (± S.E.M., n=4 independent protein
isolations from different animals). Data was analyzed using a one-way analysis of variance with
a post hoc Tukey’s test (p<0.05); for each parameter measured, values that are not statistically
different from each other share the same letter notation.
49
Figure 3.5: Changes in binding of the transcription factor NFATc1, NFATc3, and NFATc4 to a
DNA-binding element designed for the NFAT consensus sequence in the skeletal muscle of I.
tridecemlineatus over the torpor-arousal cycle. DNA-Protein Interaction (DPI)-ELISA
absorbance readings were corrected by subtraction of negative controls containing no protein and
values were expressed relative to EC. Histograms show mean relative values ± S.E.M., n=4
independent biological replicates for each of the seven experimental conditions (ER, EC, EN,
ET, LT, EA, LA). Refer to Figure 3.2 and the Materials and Methods section for more
information about the seven sampling points.
50
Figure 3.6: Effect of adjusting temperature on transcription factor-DNA binding of NFATc1, c3,
and c4. (a) NFAT-DNA binding was measured at 37, 24 (room temperature), and 4ºC at the EC
sampling point before hibernation is initiated. (b) NFAT-DNA binding was measured at 37, 24
(room temperature), and 4ºC at the LT sampling point deep within hibernation. (c) Changes in
the binding of NFAT c1, c3, and c4 transcription factors to DNA at physiological temperatures
from EC (37ºC) and LT (4ºC). Modified DPI-ELISA absorbance readings were corrected by
subtraction of no protein controls, and values were expressed relative to 37ºC.
51
Figure 3.7: Effect of adding free urea and Ca2+ on transcription factor-DNA binding of
NFATc1, c3, and c4. (a) Transcription factor-DNA binding was measured during the LT
sampling point with no urea added (control), 5mM Urea added, and 100mM Urea added. (b)
Transcription factor-DNA binding was measured during the LT sampling point with no Ca2+
added, 100nM Ca2+ added, and 600nM Ca2+ added. Modified DPI-ELISA absorbance readings
were corrected by subtraction of negative control containing no protein, and values were
expressed relative to the control (no Ca2+ or no urea added).
52
Chapter 4
Nuclear factor of activated T cells regulates cardiac hypertrophy
through calcium signaling during hibernation
53
Introduction
As discussed in Chapters 1 and 3, each organ/tissue of the I. tridecemlineatus must make
specific adjustments that allow them to maintain or readjust physiological function at low Tb in
order to survive torpor, and hibernation in general. Another tissue that undergoes physiological
adaptations is the heart. As discussed in chapter 1, heart rate is reduced strongly during torpor,
often to just 5-10 beats/min in comparison with the euthermic rates of 350-400 beats/min; this in
addition to the increased viscosity of blood at low Tb values required changes in cardiac
dynamics (Frerichs & Hallenbeck, 1998; Frerichs et al., 1994). As a result, the strength of each
individual contraction must be significantly greater and as a result cardiomyocyte hypertrophy is
observed (Nakipova et al., 2007; Storey & Storey, 2004; Wickler et al., 1991). This adaptation is
accompanied by enhanced protein synthesis, adjustments to the structure of sarcomeres, as well
as changes in gene transcription (Frey, Katus, Olson, & Hill, 2004; Li et al., 2013; Nelson &
Rourke, 2013; Tessier & Storey, 2012). Interestingly, hibernators are able to undergo reversible
cardiac hypertrophy, where the heart hypertrophies during hibernation but this process is
reversed while coming out of hibernation, and this may be related to molecular adaptations that
are just beginning to be discovered (Fahlman et al., 2000; Nelson & Rourke, 2013; Tessier &
Storey, 2012; Yan, Kudej, Vatner, & Vatner, 2015). Understanding how these animals can
rapidly and effectively reverse cardiac hypertrophy would provide novel insight into
understanding and perhaps treating cardiomyopathies and heart failure.
The present chapter focuses again on the role of calcium signaling in regulating the
NFAT family of transcription factors and their downstream targets, which are suspected to play a
crucial role in cardiac muscle remodeling. NFATs may contribute to the adaptive responses by
all muscle cell types in the body, including those needed to remodel cardiac muscles during
54
hibernation. Cardiac expression of NFAT has been shown to regulate cardiomyocyte atrophy,
apoptosis, development, and growth (Li et al., 2013; Lin et al., 2009; Liu et al., 2006; Molkentin
et al., 1998; Schubert et al., 2003). With regard to hibernation, the use of isobaric tag for relative
and absolute quantification (iTRAQ) technology with cardiac tissue of hibernating woodchucks
(Marmota monax) demonstrated an up-regulation of the NFAT pathway during hibernation.
Furthermore, expression of constitutively-active calcineurin or NFATc4 in cardiac tissue of
transgenic mice induced cardiac hypertrophy (Molkentin et al., 1998). As discussed in Chapter 3,
NFATs are activated via dephosphorylation by calcineurin, a calmodulin-stimulated protein
phosphatase, allowing NFATs to translocate to the nucleus and bind to transcriptional complexes
of their target genes (Rusnak & Mertz, 2000). Calmodulin (CAM) is a calcium-binding protein
that controls various signaling pathways, including the NFAT-calcineurin pathway. Another
important protein that regulates the NFAT-calcineurin pathway is the calcium-dependent
protease calpain. μ-calpain (calpain1) causes proteolysis of the auto-inhibitory domain of
calcineurin, which causes it to translocate to the nucleus and become constitutively active
(Burkard, 2005; Lee et al., 2014; Shioda et al., 2006; Wu et al., 2004). Therefore, calpain and
CAM both play vital roles in regulation of the NFAT-calcineurin pathway and NFATs may
contribute to the adaptive responses needed for cardiac muscle remodeling during hibernation
(Figure 1.3).
Considering the unique tendencies of hibernators to undergo reversible cardiac
hypertrophy by remodeling their cardiac muscle over the course of the hibernation season, we
hypothesized that Ca2+ signaling regulation of the NFAT-calcineurin pathway would play a
significant role in this process, particularly by modulating the gene expression of selected
proteins fundamental to muscle design. As discussed in chapter 3, myoferlin is one such protein
55
that is highly expressed in skeletal muscle and to a lesser degree in cardiac muscle (Davis et al.,
2000). It contains multiple NFAT-binding sites in its promoter, which drives high levels of
myoferlin expression in vivo and in vitro, allowing it to act as a muscle membrane protein that
promotes hypertrophy (Davis et al., 2002; Doherty et al., 2005). Similarly, myomaker is another
a membrane protein found in muscle that appears to play an essential role in muscle
development, and we demonstrated a novel finding in the previous chapter that the promoter of
myomaker contains NFAT-binding domains as well (Millay et al., 2013). The present chapter
analyzed the protein expression of calcineurin, calmodulin, calpain, NFATc1-4, myoferlin, and
myomaker over the torpor-arousal cycle in the cardiac muscle of thirteen-lined ground squirrels.
Materials and Methods
Animals
All animals were captured, treated, and their tissues were harvested following the same
protocol as previously described in Chapter 2. The cardiac tissue used was a mixture of atrial and
ventricular tissue.
Total Protein Extract Preparations
Samples of frozen cardiac muscle (approximately 500 mg) were homogenized for total
protein extraction as previously described. Total protein extracts were used for western blotting
experiments as previously described in Chapter 2.
Western Blotting
Western blotting was performed as described in Chapter 2 and 3 for six time points (EC,
EN, ET, LT, EA, IA) for NFATc1-4, myoferlin, and myomaker, calcineurin, CAM, and
56
calpain1. Equal amounts of protein from each sample (25 μg) were loaded onto 6% (NFATc1-4,
myoferlin), 8% (calcineurin, calpain1), or 15% (myomaker, CAM) polyacrylamide gels and were
run at 180 V for 60-120 min. For calmodulin, 35 µg of protein for each sample were loaded on
the polyacrylamide gels. Proteins were then transferred to PVDF membranes by electroblotting
at 320 mA for 60 min (myomaker), 90 min (calcineurin, calpain1), 120 min (NFATc1-3,
myoferlin), or at 30 V for 100 min (calmodulin). Membranes incubated in primary antibodies
were blocked for 30 min with 2.5 (for NFATc1-4) or 5% (for CnA, CAM, calpain1, myoferlin
and myomaker) w:v milk in 1x TBST.
Antibodies specific for mammalian NFATc1-4, myoferlin, and myomaker, calcineurin,
CAM, and calpain1 were provided by the manufacturers indicated in Chapter 3. NFATc1-4,
myoferlin, and myomaker antibodies were used at a 1:500 v:v dilution in 1x TBST. All other
antibodies were used at a 1:1000 v:v dilution in 1x TBST. All primary antibody incubations took
place over one night. Membranes that had been probed with myomaker (TMEM8c) were
incubated with HRP-linked anti-goat IgG secondary antibody (BioShop: 1:6000 v:v dilution). All
other antibodies were detected using HRP-linked anti-rabbit IgG secondary antibody (Bioshop:
1:6000 v:v dilution). All secondary antibody incubations were 30 minutes. The primary
antibodies cross-reacted with a single band on immunoblots at molecular weights indicated in
Chapter 3. Other parts of the procedure were carried out in accordance with the protocol
described in Chapter 2.
Quantification and Statistics
Band densities for NFATc1-4, myoferlin, myomaker, calcineurin, CAM, and calpain-1
on chemiluminescent immunoblots were visualized and quantified as described in Chapter 2.
Data are expressed as means ± SEM, n=4 independent samples.
57
Results
Analysis of NFATc1-4 protein levels in cardiac muscle
Analysis was performed on NFAT c1-4 protein levels by comparing immunoblots of
cardiac muscle from six sampling points on the torpor-arousal cycle: EC, EN, ET, LT, EA, IA
(Figure 4.1). In cardiac tissue, NFATc1 levels did not change significantly over the course of the
torpor-arousal time course. On the other hand, NFATc2 levels increased during torpor, peaking
at ET (1.88-fold increase from EN, p<0.05). NFATc3 protein levels were unchanged over the
torpor-arousal cycle. Lastly, NFATc4 levels remained stable over torpor, but increased during
arousal, peaking at IA (1.66 fold higher than ET, p<0.05) (Figure 4.1).
Analysis of myoferlin and myomaker (TMEM8c) protein levels
Similar to skeletal muscle in Chapter 3 (Figure 3.3), myoferlin levels in cardiac muscle rose
dramatically at ET (2.68 fold higher in comparison with control, p<0.05). In contrast to skeletal
muscle levels, myomaker in cardiac muscle also peaked at ET (1.88 fold higher in comparison
with control, p<0.05) (Figure 4.2).
Analysis of calcineurin A, calmodulin, and calpain1 protein levels in cardiac muscle
CnA protein levels remained fairly stable throughout the torpor-arousal cycle, with the
exception of IA, where levels decreased dramatically by 62% from EA (p<0.05). Calmodulin
levels were highest at EC and EN, but levels began to decrease dramatically upon entering
arousal, with the lowest levels being observed at EA (70% decrease in comparison with EC,
p<0.05). Calpain levels showed a similar trend, with the highest levels being observed at EC and
IA, and drastically lower levels throughout the torpor-arousal cycle. The lowest levels were
observed during EN (85% decrease from EC, p<0.05) (Figure 4.3).
58
Discussion
The current chapter aimed at furthering our understanding of the molecular mechanisms
underlying cardiac muscle remodeling during hibernation in the 13-lined ground squirrel. As
discussed in chapter 1, I. tridecemlineatus is an excellent animal model for studying cardiac
muscle remodeling with applications for the treatment of cardiac hypertrophy and heart failure
because its cardiac muscle is known to naturally undergo reversible cardiac hypertrophy (Li et
al., 2013; Nelson & Rourke, 2013). However, the molecular mechanisms that are responsible the
changes of muscle structure/function during hibernation are not well known. Therefore, the
present study focuses on the family of transcription factors known as NFATs, which have been
shown to regulate targets associated with cardiomyocyte hypertrophy, apoptosis, and
development (Li et al., 2013; Lin et al., 2009; Liu et al., 2006; Molkentin et al., 1998; Tessier &
Storey, 2012).
NFATs are regulated by calcineurin, a calmodulin-stimulated phosphatase that
dephosphorylates NFATs, thus allowing them to translocate into the nucleus (Rusnak & Mertz,
2000). Calcineurin is sensitive to changes in calcium levels and it is positively regulated by other
calcium-signaling proteins such as CAM and calpain (Al-Shanti & Stewart, 2009; Burkard,
2005; Lee et al., 2014; Shibasaki et al., 2002; Shioda et al., 2006; Wu et al., 2004; Yang & Klee,
2000). Therefore, experiments were conducted to study the roles of calcineurin, CAM, and
calpain as upstream regulators of the NFAT pathway within the context of reversible cardiac
hypertrophy during hibernation. Furthermore, the downstream targets myoferlin and myomaker
were evaluated because of their newly identified and crucial role as muscle membrane proteins in
muscle development and repair (Demonbreun et al., 2010; Doherty et al., 2005; Millay et al.,
2013, 2014).
59
In cardiac muscle, the response of NFATc1-4 transcription factors as well as the
downstream targets myoferlin and myomaker were enhanced during torpor (Figures 4.1, 4.2).
Similarly to skeletal muscle, NFATc2 was significantly elevated during torpor. Although
myoferlin was elevated in both heart and skeletal muscle, myomaker remained stable in skeletal
muscle throughout hibernation. In the heart, myomaker showed a spike at ET (1.88 fold higher in
comparison with control, p<0.05) (Figure 4.2). The regulator of NFATs, calcineurin decreased
during arousal by 62% from EA to IA (p<0.05). One of the activators of calcineurin, CAM, also
decreased during LT and throughout arousal. Similarly, calpain levels declined from EC to EA
by 61% (p<0.05). Calpain cleaves the autoinhibitory domain of calcineurin, thus activating it
(Burkard, 2005; Lee et al., 2014; Shioda et al., 2006; Wu et al., 2004). Therefore, these Ca2+-
signaling proteins (CAM, and calpain) as well as the NFAT-CnA pathway and its downstream
muscle targets (myoferlin and myomaker) could be part of the mechanism regulates reversible
cardiac muscle hypertrophy. During torpor, NFATc2 could be causing upregulation of myoferlin,
thus promoting cardiac hypertrophy to increase contractility to the heart in response to the
decrease in heart rate during hibernation (Frerichs & Hallenbeck, 1998; Frerichs et al., 1994).
Then, the mechanism behind the rapid reduction of cardiac mass may be initiated by decreases in
calcineurin activity, which is the result of lower calpain and CAM levels upon and before
entering arousal. Subsequently, NFATc2 levels decrease and this may be causing the
downregulation of both myoferlin and myomaker, thus reversing the cardiac hypertrophic
stimulus. Although the NFAT-calcineurin pathway has been studied in association with cardiac
hypertrophy in the past, the present findings identify a novel mechanism of rapidly naturally
reversing cardiac hypertrophy, whereby decreased calcium signaling results in a downregulation
60
of muscle hypertrophy proteins targeted by NFATs (Li et al., 2013; Lin et al., 2009; Molkentin et
al., 1998).
In summary, the present chapter provides insight into some of the key proteins involved
in cardiac muscle remodeling during hibernation. Although more remains to be investigated,
including the regulation of factors associated with muscle atrophy and the reduction in cardiac
dimensions (Chapter 6), the results from this chapter indicate that the Ca2+ signaling proteins
(calcineurin, calmodulin, calpain) regulating the activity of NFATc1-4. as well as their
downstream targets, myoferlin and myomaker, are important to the hypertrophy of cardiac
muscle during hibernation. This is also the first study that has found the myomaker protein to be
expressed in the heart, although its specific role still remains unclear. Further elucidation of the
role that the Calcineurin-NFAT pathway plays in reversible cardiac hypertrophy in thirteen-lined
ground squirrels will not only contribute to our understanding of cardiac remodeling, it could
potentially lead to the applications for treating maladaptive cardiac hypertrophy and heart failure.
Specifically, the identification of calpain and calmodulin as initiators for the reversal of cardiac
hypertrophy in squirrels shows promise. The unique and tissue-specific mechanisms in ground
squirrel of preserving skeletal muscle (Chapter 3) and undergoing reversible cardiac hypertrophy
during the torpor-arousal cycle make studying this animal biologically and clinically-relevant.
61
Figures
Figure 4.1: Changes in the protein levels of NFAT transcription factors over the course of the
torpor-arousal cycle in cardiac muscle of I. tridecemlineatus. NFATc1, c2, c3, and c4 total
protein expression levels were visualized at six sampling points: EC, EN, ET, LT, EA, and IA.
Representative Western blots and Coomassie total protein loading controls are shown for
selected pairs of sampling points that are labeled to the left and right of the gel. Sample numbers
(lanes) are labeled along the top indicating 4 samples of one type (e.g., EN lanes 1, 2, 3, 4) and 4
samples of another (e.g. LT lanes 5, 6, 7, 8) using NFATc2 as an example. Also shown are
histograms with mean standardized band densities (± S.E.M., n=4 independent protein isolations
from different animals). Data was analyzed using a one-way analysis of variance with a post hoc
Tukey’s test (p<0.05); for each parameter measured, values that are not statistically different
from each other share the same letter notation. Figure from (Zhang & Storey, 2015).
62
Figure 4.2: Changes in myoferlin and myomaker total protein levels in cardiac muscle over the
torpor-arousal cycle in I. tridecemlineatus. Representative Western blots and Coomassie total
protein loading controls are shown for selected pairs of sampling points that are labeled to the
left and right of the gel. Sample numbers (lanes) are labeled along the top indicating 4 samples of
one type (e.g., EC lanes 1, 2, 3, 4) and 4 samples of another (e.g. EN lanes 5, 6, 7, 8) using
Myoferlin as an example. Also shown are histograms with mean standardized band densities (±
S.E.M., n=4 independent protein isolations from different animals). Data was analyzed using a
one-way analysis of variance with a post hoc Tukey’s test (p<0.05); for each parameter
measured, values that are not statistically different from each other share the same letter notation.
Figure from (Zhang & Storey, 2015).
63
Figure 4.3: Changes in calcineurin, calmodulin, and calpain total protein levels in cardiac
muscle over the torpor-arousal cycle in I. tridecemlineatus. Representative Western blots and
Coomassie total protein loading controls are shown for selected pairs of sampling points that are
labeled to the left and right of the gel. Sample numbers (lanes) are labeled along the top
indicating 4 samples of one type (e.g., EC lanes 1, 2, 3, 4) and 4 samples of another (e.g. EN
lanes 5, 6, 7, 8) using Calmodulin as an example. Also shown are histograms with mean
standardized band densities (± S.E.M., n=4 independent protein isolations from different
animals). Data was analyzed using a one-way analysis of variance with a post hoc Tukey’s test
(p<0.05); for each parameter measured, values that are not statistically different from each other
share the same letter notation. Figure from (Zhang & Storey, 2015).
64
Chapter 5
Regulation of Foxo4 and MyoG promotes skeletal muscle atrophy
during torpor in ground squirrels
65
Introduction
As discussed in Chapter 1, I. tridecemlineatus needs to make significant adaptations in
order to survive the periods of low Tb during torpor, the fluctuations in Tb as a result of the
torpor-arousal cycles, and for bodily functions to be restored following hibernation so that it can
resume natural activities and scavenge for food. In order to resume post-hibernation activities,
hibernators need to avoid significant disuse-induced skeletal muscle atrophy (muscle protein
degradation) during hibernation, otherwise muscle mass, strength, and relative amount of
oxidative muscle fibers will all be reduced (Bassel-Duby & Olson, 2006; Choi et al., 2009;
Malatesta et al., 2009; Rourke et al., 2004). Interestingly, it was demonstrated that the relative
ratio of muscle mass/body weight actually increases throughout hibernation in ground squirrels
due to significant losses in body weight as well as an extremely effective mechanism of muscle
preservation and remodeling that is unique to hibernators (Gao et al., 2012). Therefore,
uncovering the molecular mechanisms underlying this process of muscle maintenance, that
occurs naturally, have clinical relevance for therapeutic intervention of muscle wasting and
assisting in the physical rehabilitation.
Recent studies have begun to elucidate the molecular basis of muscle remodeling and
preservation, with findings indicating that the peroxisome proliferator-activated receptor γ
coactivator 1-α (PGC-1α) and the NFAT family of transcription factors are implicated in this
process (Xu et al., 2013; Zhang & Storey, 2015). However, a current gap in knowledge exists
around whether the molecular pathways of muscle atrophy and protein degradation are still
activated during hibernation as a result of inactivity. It is important to understand whether
muscle remodeling and hypertrophy pathways involving PGC-1α and NFAT simply balance the
66
muscle wasting pathways to maintain muscle mass, or whether there is an inhibition of signaling
pathways that promote muscle atrophy, which would otherwise be activated with prolonged
inactivity in non-hibernators. Addressing this question is important for the development of
therapies, and whether they should inhibit muscle atrophy signaling pathways or promote muscle
remodeling and hypertrophy, or attempt to do both, in order to treat muscle wasting.
As discussed in Chapter 1, the main signaling pathway that controls muscle atrophy
involves the forkhead box transcription factors of the O subclass (Foxo) as well as the myogenin
(MyoG) transcription factor, and their regulation of the ubiquitin proteasome system (UPS)
(Moresi et al., 2010; Sandri et al., 2004; Stitt et al., 2004). The UPS is an important mechanism
for protein degradation, whereby substrates are ligated to ubiquitin via E3 ubiquitin ligases like
Muscle Atrophy F-Box (MAFbx/atrogin-1) and Muscle Ring Finger 1 (MURF1), which target
these substrates for degradation in the proteasome (Foletta et al., 2011; Herrmann et al., 2007;
Schiaffino et al., 2013). Due to the importance of both MAFbx and MURF1 for muscle atrophy,
common regulators were found for both ligases. The Foxo family of transcription factors were
the first of such factors (Moresi et al., 2010; Sandri et al., 2004; Stitt et al., 2004). Then, MyoG,
which was initially identified as a regulator of myogenesis, was shown to be a positive regulator
of both E3 ligases as well; where the expression of MAFbx and MURF1, as well as muscle
atrophy were attenuated in MyoG-null mice (Moresi et al., 2010). The mammalian Foxo family
has four members: Foxo1, Foxo3a, Foxo4, and Foxo6, that are involved in various cellular
processes in addition to muscle atrophy, such as antioxidant defense and apoptosis (Birkenkamp
& Coffer, 2003; Greer & Brunet, 2005; Wu & Storey, 2014). Foxo1, Foxo3a, and Foxo4 are all
regulated by the Akt/protein kinase B (PKB) signaling pathway (Figure 1.4). Specifically, Akt
blocks the function of all three Foxo proteins through phosphorylation at conserved residues that
67
lead to cytoplasmic localization of Foxo (Brunet et al., 1999; Matsuzaki et al., 2005; Takaishi et
al., 1999; Tang et al., 1999). For Foxo4, Akt inhibits the nuclear translocation and activation of
Foxo4 by phosphorylating the Threonine (Thr)-32, Serine (Ser)-197, and Ser262 residues
(Matsuzaki et al., 2005; Takaishi et al., 1999). However, Foxo4 transcriptional activity has been
shown to be regulated through a separate pathway, involving the Ras and Ral GTPases, as well
(De Ruiter et al., 2001; Essers et al., 2004; Kops et al., 1999; Van Den Berg et al., 2013).
Ras and the Ral isoforms, RalA and RalB, are small GTPases that share very similar
sequences, with Ral activation requiring the activation of Ras in order to act as signaling
molecules for a variety of downstream processes like transcription, DNA synthesis, and
differentiation (De Ruiter et al., 2001; Feig, Urano, & Cantor, 1996). Ral binding protein 1
(Ralbp1) is another integral part of the Ras-Ral pathway as it acts as a downstream effector of
Ral and associates with Ral in a GTP-dependent manner (Cantor, Urano, & Feig, 1995; Jullien-
Flores et al., 1995). Initially it was discovered that Foxo4 transcriptional activity was dependent
upon activation of the Ras-Ral pathway as it results in phosphorylation of Foxo4 at Thr-447 and
Thr-451 (De Ruiter et al., 2001; Kops et al., 1999) (Figure 1.5). As mentioned previously, this
pathway acts independently from the control of nuclear-cytoplasmic distribution that Akt holds
over Foxo4, as another downstream kinase that phosphorylates Foxo4 was found to be Stress-
activated protein kinase (SAPK)/Jun amino-terminal kinase 1 (JNK1). JNK1 is bound to the
cellular scaffold protein, c-Jun-amino-terminal-interacting protein 1 (JIP1), where it is activated
by RalA through phosphorylation (Van Den Berg et al., 2013) (Figure 1.5). It was demonstrated
that the activation of Foxo4 transcriptional activity through the Ras-Ral pathway and JNK was
induced by tumor necrosis factor α (TNFα)-activation of the Ras-Ral pathway (Essers et al.,
2004; Van Den Berg et al., 2013). Furthermore, the induction in MAFbx expression by TNFα is
68
reliant on Foxo4 regulation and not Foxo1/3a (Moylan, Smith, Chambers, McLoughlin, & Reid,
2008). These previous findings stress the importance of understanding the regulation of Foxo4
through both the Ras-Ral and Akt pathways, and the importance of studying how this contributes
to the control of muscle atrophy by Foxo4.
Given the unique ability of I. tridecemlineatus to avoid disuse-induced muscle wasting
despite being inactive during periods of torpor and arousal during hibernation, there is a need to
study the molecular mechanisms underlying muscle atrophy during hibernation in this animal. It
is hypothesized that during the avoidance of muscle loss during hibernation, there will be a
downregulation of MyoG- and Foxo4-mediated MAFbx and MURF1 expression, and this effect
will be initiated by the Foxo4 dephosphorylation through inhibition of the Ras-Ral pathway. To
test this hypothesis, the present study characterized the protein levels of total Foxo4 as well as
different phosphorylated forms of Foxo4, in addition to Ras, RalA, Ralbp1, MyoG, MAFbx, and
MURF1 over cycles of torpor and arousal in the skeletal muscles of I. tridecemlineatus.
Materials and Methods
Animal treatment
All animals were captured, treated and their tissues were harvested following the same
protocol as previously described in Chapter 2. The skeletal muscle used was a mixture of several
hind limb muscles.
Total protein isolation
69
Total protein extracts were prepared as previously described in Chapter 2. Samples of
frozen skeletal muscle (n=4) weighing approximately 0.5g were used to prepare total protein
extracts, which were used for western blotting. Other information as in Chapter 2.
Western blotting
Western blotting was performed as described in Chapter 2 for six time points (EC, EN,
ET, LT, EA, LA) for all the proteins studied in this chapter. Equal amounts of protein from each
sample (25 μg) were loaded onto 8% (Foxo4, p-Foxo4 S197, p-Foxo4 T451, Ralbp1, MURF1),
10% (MyoG, MAFbx), or 15% (Ras and RalA) polyacrylamide gels that were run at 180 V for
60-120 min. Proteins were then transferred to PVDF membranes by electroblotting at 160 mA
for 90 min (Foxo4, p-Foxo4, Ralbp1, MyoG, MAFbx, MURF1) or at 30 V for 100 min (Ras,
RalA). Membranes were blocked after transfer for 30 min with 7.5% (Ras, RalA, MAFbx,
MURF1) or 5% (Foxo4, p-Foxo4, MyoG, Ralbp1) w:v milk in 1x TBST. After washing,
membranes were probed with specific primary antibodies at 4°C overnight.
Antibodies specific for mammalian Foxo4 (CS 9472) from Cell Signaling Technology
(Danvers, MA), MAFbx/atrogin-1/FBXO32 (SC27645), MyoG (SC576) and p-Foxo4 S197
(SC101628) from Santa Cruz Biotechnology (Dallas, TX), MURF1/TRIM63 (GTX110475), Ras
(GTX132480) and RalA (GTX114204) from Genetex (Irving, CA), as well as p-Foxo4 T451
(12053) and Ralbp1 (38202) from Signalway Antibody (Baltimore, MD) were purchased and
used at a 1:1000 v:v dilution in 1x TBST. All membranes were incubated with HRP-linked anti-
rabbit IgG secondary antibody (Bioshop: 1:6000 v:v dilution) for 30 min at room temperature.
70
The antibodies cross-reacted with a single band on immunoblots at the expected molecular mass
for the respective proteins as indicated on the antibody specification sheets.
Data and Statistical Analysis
Band densities for Foxo4, p-Foxo4, MAFbx, MURF1, Ras, Ral, and Ralbp1 on
chemiluminescent immunoblots were visualized and quantified as described in Chapter 2. Data is
expressed as mean ± SEM with n=4 independent samples from different animals.
Results
Upregulation and activation of Foxo4 at various stages during the torpor-arousal cycle
To determine the role of Foxo4 transcription factors in skeletal muscle remodeling during
the torpor-arousal cycle, total expression of Foxo4 as well as relative phosphorylation of its Ser-
197 and Thr-451 residues was determined via western blotting at six different sampling points:
EC, EN, ET, LT, EA, LA. As shown in Figure 5.1, Foxo4 protein levels increased dramatically
upon entering torpor at EN (2.14-fold increase compared to EC, p<0.05). Foxo4 levels continued
to increase until LT, where levels were 3.9-fold higher than EC (p<0.05). Following LT, protein
levels began to decline progressively throughout arousal. Two phosphorylated residues of Foxo4
were analyzed as well, Ser-197 and Thr-451. The Thr-451 residue of Foxo4 on its C terminus is
known to be phosphorylated by JNK through activation of the Ras-Ral pathway, whereas Ser-
197 is phosphorylated by Akt/PKB, leading to inhibition of Foxo4 (De Ruiter et al., 2001; Essers
et al., 2004; Matsuzaki et al., 2005). We observed differential phosphorylation of the two
residues over the torpor-arousal cycle; where p-Foxo4 T451 levels increased dramatically upon
71
entering torpor by 1.68-fold compared to EC (p<0.05). However, protein levels declined quickly
as the squirrels progressed through torpor, where there was a 44% decrease at ET compared to
EC (p<0.05). p-Foxo4 T451 levels then remained low throughout the torpor-arousal cycle
(Figure 5.1). On the contrary, p-Foxo4 S197 residue levels decreased immediately upon entering
torpor with EN levels decreasing by 70% in comparison with EC (p<0.05). p-Foxo4 S197 levels
remained low during torpor, but returned to euthermic levels during arousal (Figure 5.1).
Analysis of p-Foxo4 vs. total Foxo4 ratios
Other than analyzing protein levels of Foxo4 and two of its phosphorylated forms relative
to the total protein loading control, phosphorylation ratios were calculated from the ratio of p-
Foxo to total Foxo levels. The way of interpreting the data takes changes in total protein levels
into account when analyzing the changes in phosphorylation. As seen in Figure 5.2, the ratio of
p-Foxo4 to total Foxo4 for the Thr-451 residue showed a progressive decrease after entering
torpor, with a 77% decrease being observed at ET relative to EC (p<0.05). At LT, there was a
significant rise in phosphorylation by 2-99-fold relative to ET (p<0.05) and the ratio remained
stable after entering arousal. Similar to p-Foxo4 protein levels, the phosphorylation ratios for the
Ser-197 residue differed from those of the Thr-451 residue. p-Foxo4 S197 ratios declined
dramatically immediately upon entering torpor, as there was a 88% decrease in EN relative to EC
(p<0.05). These levels remained relatively stable throughout the rest of the torpor-arousal cycle,
with increases occurring at ET and during arousal (both EA and LA) (Figure 5.2). However,
these changes were not significant.
Analysis of MyoG, MAFbx, and MURF1 protein levels
72
MyoG protein levels were analyzed and we observed that it remained relative stable
throughout the torpor-arousal cycle with the exception of LT, where MyoG levels declined by
52% relative to EC (p<0.05) (Figure 5.3). MAFbx levels declined progressively upon entering
torpor, with a significant decrease occurring at ET (42% decrease relative to EC, p<0.05) and
again at LT (69% decline relative to EC, p<0.05). Afterwards, MAFbx returned to similar levels
as ET during arousal (Figure 5.3). MURF1 showed different expression patterns from MAFbx
even though they are both E3 ubiquitin ligases that target proteins for degradation (Foletta et al.,
2011; Lecker, 2003; Schiaffino et al., 2013). MURF1 levels also declined during ET by 40% in
comparison with EC (p<0.05). However, there was a 2.66-fold increase at LT relative to ET
(p<0.05). This is spike in MURF1 levels was followed by another significant decline by 60%
from LT to EA (p<0.05). During late arousal, protein levels returned to euthermic levels (Figure
5.3). The MAFbx timecourse western blots were done by Dr. Shannon N. Tessier and are
unpublished and not part of her thesis, she contributed her data on this target as it was vital to the
present chapter and she is a co-author on our submitted manuscript for publication.
Analysis of the Ras-Ral pathway
Ras-Ral signaling has been identified as a regulator of Foxo4 through phosphorylation,
and it involves two small GTPases (Ras and Ral) and possibly Ralbp1 (De Ruiter et al., 2001;
Essers et al., 2004; Neel et al., 2011). The protein expression of the three above mentioned
proteins were characterized. As shown on Figure 5.4, we observed a dramatic decline in Ras
immediately upon entering torpor, with levels declining by 93% from EC to EN (p<0.05). These
levels remained stable throughout the torpor-arousal cycle. On the other hand, RalA levels
increased significantly at EN from EC by 1.65-fold (p<0.05). Afterwards, protein expression
73
decreased significantly at ET by 64% relative to EN (p<0.05), and these levels remained
relatively stable over the rest of the torpor-arousal cycle (Figure 5.4). Ralbp1 expression
increased dramatically as well upon entering torpor by 2.61-fold relative to EC (p<0.05). At LT,
there was a significant decrease in protein levels back to euthermic levels (54% relative to EC,
p<0.05). Afterwards, Ralbp1 expression increased again during arousal, with a 2.33-fold increase
being observed at EA relative to LT (p<0.05) (Figure 5.4)
Discussion
The present study furthers our understanding of the molecular basis behind muscle
remodeling and preservation during hibernation in I. tridecemlineatus. Previous studies have
shown that during hibernation, pathways that promote muscle hypertrophy and muscle fiber
shifting towards oxidative slow fibers are active, contributing to the preservation of total muscle
mass and fiber typing (Xu et al., 2013; Zhang & Storey, 2015). Therefore, we sought to address
the question of whether molecular pathways that promote muscle atrophy are inhibited during
hibernation, or if they are still upregulated as a result of inactivity but are balanced by the above
mentioned pathways. Due to the role of the Foxo family of transcription factors and MyoG as
central regulators of muscle atrophy via the UPS, we characterized the expression MyoG, Foxo4,
as well as MAFbx and MURF1; the E3 ubiquitin ligases that are regulated by these transcription
factors (Moresi et al., 2010; Sandri et al., 2004; Stitt et al., 2004). Previous work has shown that
total Foxo3a protein levels as well as levels of phosphorylated Foxo3a (p-Foxo3a) at Serine 253
(S253) are upregulated throughout the torpor-arousal cycle, peaking at LT (Wu & Storey, 2014).
When taking into consideration the ratio of p-Foxo3a/total Foxo3a, there is actually no change in
the phosphorylation status of Foxo3a. Also, phosphorylation of Foxo3a at the Ser-253 residue
74
inhibits Foxo3a from translocating to the nucleus and regulating gene expression (Brunet et al.,
1999; Dobson et al., 2011). Therefore, the increase in p-Foxo3a at the Ser-253 residue suggests
that although there is upregulation of Foxo protein levels, most if not all of these additional
Foxo3a proteins are inactive. Therefore, there is a need to further study the signaling pathways
regulating muscle atrophy during hibernation, specifically ubiquitin ligases regulated by Foxo
transcription factors.
In this study, protein level analysis was conducted on MAFbx, MURF1, Foxo4, and
MyoG in addition to the main factors of the Ras-Ral pathway; including Ras, RalA, and Ralbp1.
After studying the UPS, it was found that during torpor there were decreased protein levels of
both MAFbx and MURF1, two E3 ubiquitin ligases that are regulated by Foxo1, 3a, 4, and are
essential to muscle atrophy (Sandri et al., 2004; Stitt et al., 2004; Waddell et al., 2008). MAFbx
protein levels progressively declined through torpor, with the greatest decrease occurring at LT
(69% relative to EC, p<0.05). MURF1 levels also decreased at ET by 40% compared to EC
(p<0.05) but its levels rose dramatically at LT (Figure 5.3), suggesting that these two ligases
could be differentially regulated by the same or different transcription factors. One such
transcription factor that is known to regulate the expression of both ligases is MyoG, and its
pattern of protein expression resembles that of MAFbx during the torpor-arousal cycle, with a
significant decrease in MyoG levels occurring at LT (Figure 5.3) (Bricceno et al., 2012;
MacPherson, Wang, & Goldman, 2011; Moresi et al., 2010). It has been observed that inhibiting
MyoG through the use of trichostatin A results in reduced muscle wasting, and there is a greater
decline in MAFbx expression in comparison with MURF1 expression, suggesting that MyoG
may regulate the expression of MAFbx to a greater extent than MURF1 Bricceno et al., 2012).
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Another important regulator of both MAFbx and MURF1 is Foxo4, and total levels of
this protein significantly increased from EC and remained elevated throughout the torpor-arousal
cycle, peaking at LT, where there was a 3.9-fold increase in comparison with EC (p<0.05)
(Figure 5.1). p-Foxo4 levels were also assessed, and we found differential expression of the two
p-Foxo4 residues, Serine 197 (S197) and Threonine 451 (Thr451). p-Foxo4 S197 protein levels
decreased by 70% while entering torpor (EN) in comparison with EC (p<0.05). On the other
hand, at the same time point, p-Foxo4 T451 levels increased by 1.68-fold relative to EC
(p<0.05). The ratios of p-Foxo4 to total Foxo4 were also calculated in order to accurately assess
Foxo4 activity and phosphorylation status; taking into account changes in total protein levels
(Zhang et al., 2010). The results indicate that there are differences in phosphorylation status
between the two residues as well, with p-Foxo S197 showing decreased phosphorylation
throughout torpor. However, a decrease in phosphorylation status is only seen at ET for p-Foxo4
T451 (77% decrease relative to EC, p<0.05) (Figure 5.2).
The explanation for the differential phosphorylation at these residues is that the Ser-197
residue is phosphorylated by Akt/PKB, a commonly known inhibitor of Foxo4, whereas the Thr-
451 residue is phosphorylated by JNK1 through the Ras-Ral pathway, resulting in transcriptional
activation of Foxo4 (Figure 1.5) (De Ruiter et al., 2001; Essers et al., 2004; Kops et al., 1999;
Matsuzaki et al., 2005; Takaishi et al., 1999). Findings from this study indicated that the Ras
protein declined immediately upon entering torpor, whereas RalA and its effector protein,
Ralbp1, were initially upregulated during EN, then levels of both proteins progressively declined
throughout torpor, reaching levels that were 41% and 54% that of EC for RalA and Ralbp1,
respectively (Figure 5.4). Furthermore, this progressive downregulation of the Ral pathway
during torpor mirrors the decline in activated JNK, or p-JNK1 (Thr-183/Tyr-185), levels
76
throughout torpor, which was found in previous work from our lab (Wu & Storey, 2014). As
mentioned previously, active RalA leads to phosphorylation and activation of JNK1 that is bound
to JIP1. This occurs through the accumulation of reactive oxygen species (ROS), resulting in
TNFα-mediated activation of the Ras-Ral pathway (Essers et al., 2004; Van Den Berg et al.,
2013). Given that in I. tridecemlineatus, ROS production is positively associated with Tb
changes, this explains why the Ras-Ral pathway and p-JNK levels decline over torpor, as Tb
continues to decrease (Brown, Chung, Belgrave, & Staples, 2012).
In summary, these results indicate that although there are significant increases in total
Foxo4 protein levels throughout torpor and early arousal, the ratio of dephosphorylated Foxo4
(S197) able to translocate to the nucleus and regulate transcription, is significantly decreased.
Also, since the phosphorylation ratio of p-Foxo4 (T451) is significantly decreased at ET due to
inactivation of the Ras-Ral pathway and JNK, the amount of active nuclear Foxo4 able to
regulate the expression of target genes like MAFbx and MURF1 is significantly reduced as well.
Therefore, we can conclude that although Foxo4 is upregulated during torpor, the reduction in
Foxo4 activity is more significant during torpor, especially at ET. This finding, along with the
downregulation of MyoG during torpor, leads to the decreased expression of MAFbx and
MURF1. This mechanism would result in reduced muscle atrophy during torpor despite
mechanical unloading, so it may contribute to the preservation of muscle mass while ground
squirrels hibernate. Therefore, in addition to upregulating pathways to promote hypertrophy and
fiber type switching, signaling pathways regulating muscle atrophy, such as the Ras-Ral
pathway, are downregulated during hibernation (Xu et al., 2013; Zhang & Storey, 2015). These
novel findings on this unique and natural physiological process in ground squirrels advances our
knowledge of skeletal muscle remodeling, and they could be applied for therapeutic intervention
77
in treating muscle wasting diseases like Spinal Muscular Atrophy and Duchenne Muscular
Dystrophy. Specifically, our findings suggest that in addition to testing agents that promote
muscle growth, like Naloxone – an NFAT activator, there needs to be an emphasis on testing
inhibitors of muscle atrophy signaling pathways. For example, farnesyltransferase inhibitors
designed to block the Ras-Ral pathway for anticancer treatment could also be tested for the
possibility of inhibiting Foxo4 and reducing muscle atrophy (Yeh & Der, 2007).
78
Figure
Figure 5.1: Changes in the protein levels of the Foxo4 transcription factors and its
phosphorylated forms, Ser-197 (S197) and Thr-451 (T451), over the course of the torpor-arousal
cycle in skeletal muscle of I. tridecemlineatus. Foxo4, p-Foxo4 S197, and T451 protein
expression levels were visualized at six sampling points: EC, EN, ET, LT, EA, LA. See Chapter
1 Materials and Methods and for more extensive definitions. Other information as in Figure 3.2.
79
Figure 5.2: Changes in phosphorylation ratios for phosphorylated Foxo4 proteins were analyzed
by taking a ratio of band densitometries between p-Foxo4 protein levels and total Foxo4 protein
levels. Phosphorylation ratios were determined for p-Foxo4 S197 and T451. Other information
as in Figure 5.1.
80
Figure 5.3: Changes in protein levels of the muscle proteins MyoG, MAFbx, and MURF1 over
the course of the torpor-arousal cycle in skeletal muscle of I. tridecemlineatus. Other information
as in Figure 3.2. The MAFbx timecourse western blots were done by Dr. Shannon N. Tessier and
are unpublished and not part of her thesis. She contributed her data on this target as it was vital to
the present chapter and she is a co-author on our submitted manuscript for publication.
81
Figure 5.4: Changes in protein levels of proteins in the Ras-Ral pathway: Ras, RalA, and Ralbp1
over the course of the torpor-arousal cycle in skeletal muscle of I. tridecemlineatus. Other
information as in Figure 3.2.
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Chapter 6
Transcriptional activation of muscle atrophy promotes cardiac muscle
remodeling during mammalian hibernation in ground squirrels
83
Introduction
As discussed in Chapters 1 and 4, each organ/tissue of the hibernating ground squirrel
must make specific adjustments that allow them to maintain or readjust physiological functions
at low Tb values to support long term torpor. Heart rate is strongly reduced during torpor, often
from euthermic rates of 350-400 beats/min to just 5-10 beats/min. These changes in heart rate,
plus the increased viscosity of blood at low Tb values, require adjustments to be made in squirrel
heart dynamics (Frerichs & Hallenbeck, 1998; Frerichs et al., 1994). For example, the strength of
each individual contraction must be significantly increased in response to the pressure and
volume overloads; as a result cardiac hypertrophy is observed (Depre et al., 2006). In most
mammals, cardiac hypertrophy is characterized by significant cardiac fibrosis whereby collagen
deposition stiffens cardiac chamber walls, reduces diastolic filling, and ultimately prevents the
heart from pumping enough blood to meet body demands; this is a condition known as heart
failure (Day, 2013). Interestingly, previous findings have shown that ground squirrels in
hibernation have larger left ventricular mass, internal dimensions, and wall dimensions in
comparison with active squirrels; suggesting reversible left ventricular hypertrophy is occurring
(Nelson & Rourke, 2013). This fascinating process may be related to molecular adaptations that
occur during hibernation, which are just beginning to be discovered. Understanding the
mechanism of reversible cardiac hypertrophy in hibernators would provide novel insight into the
development and treatment of maladaptive cardiac hypertrophy and heart failure.
As mentioned previously, one of the key elements of cardiac hypertrophy is the stress-
induced adaptation in protein turnover; which involves protein synthesis and degradation. Both
of these mechanisms are activated by increased cardiac workload due to pressure or volume
84
(Depre et al., 2006). Upregulation of NFAT has been implicated in the increased synthesis of
numerous proteins during cardiac hypertrophy as shown in Chapter 4. With regard to protein
degradation, the UPS is known to be an important mechanism whereby substrates are ligated to
ubiquitin via ubiquitin ligases and are targeted for degradation (Herrmann et al., 2007). As
discussed in Chapter 5, the specificity of the UPS is determined by E3 ubiquitin ligases that
recognize specific target proteins, which include MAFbx and MURF1. The role of the UPS
remains relatively unclear in cardiac muscle in comparison with skeletal muscle; although they
have shown upregulation in association with cardiac hypertrophy or heart failure (Depre et al.,
2006; Galasso et al., 2010). This increased in expression of ubiquitination machinery may be in
response to the increase in over protein production that accompanies hypertrophy in the heart or
in response to modified or damaged proteins that need to be degraded (Day, 2013). Therefore,
with respect to reversible cardiac hypertrophy, we suspect that the UPS will be mostly active
during late torpor or arousal, especially when coming out of hibernation as perfusion and Tb will
increase; thus reversing the hypertrophic stimulus and promoting atrophy instead.
As described in Chapter 5, early studies have shown that both MAFbx and MURF1 are
upregulated under similar atrophy-inducing conditions, suggesting that both ligases are regulated
by common TFs; Foxos were the first set of such factors to be identified (Sandri et al., 2004; Stitt
et al., 2004). For example, Foxo1 was shown to increase MAFbx or MURF1 levels by blocking
their inhibition from the IGF-1/PI3K/Akt insulin signaling pathway. Thus, it was initially
believed that Foxo1 indirectly increases the expression of MAFbx and MURF1 (Stitt et al.,
2004). However, later studies identified that Foxo TFs share consensus sequences that allow
them to bind directly to the MAFbx and MURF1 promoters (Sandri et al., 2004; Waddell et al.,
2008). In addition, it was also observed that not all Foxo family members bind equally to the
85
promoters of MAFbx and MURF1 (Waddell et al., 2008). For instance, Foxo1 activates the Foxo
binding motif; leading to MURF1 upregulation, to a greater degree than Foxo3a or 4 (Waddell et
al., 2008). Akt, otherwise known as protein kinase B (PKB), has been shown to block the
function of all three Foxo proteins through phosphorylation, leading to their containment in the
cytoplasm (Brunet et al., 1999; Takaishi et al., 1999; Tang et al., 1999). Dephosphorylation of
Foxo factors on the other hand leads to nuclear localization and growth suppression or apoptosis
(Takaishi et al., 1999). Akt has numerous phosphorylation sites on Foxo1, 3a, and 4, including
Threonine32 (Thr32) for Foxo3a, as well as Thr24 and Serine319 (Ser319) for Foxo1 (Dobson et al.,
2011) (Figure 1.4). Aside from Akt, Foxos can also be phosphorylated and inhibited by
numerous other kinases; including Jun N-terminal kinase (JNK), AMP-activated protein kinase
(AMPK), cyclin-dependent kinase (CDK), and MAPK-activated protein kinase (MK) (Huang,
Regan, Lou, Chen, & Tindall, 2006; Kress et al., 2011; van der Horst & Burgering, 2007; Yuan
et al., 2008) (Figure 1.4).
Aside from Foxos, Myogenin (MyoG) has also been identified as a positive regulator of
MAFbx and MURF1 (Figure 1.4); the expression of both ligases as well as muscle atrophy were
attenuated in MyoG-null mice (Moresi et al., 2010). Given the unique tendencies of hibernators
to undergo reversible cardiac hypertrophy over the course of the hibernation season, significant
cardiac remodeling involving protein turnover is believed to occur. We hypothesize that the Foxo
and MyoG TFs in addition to the E3 ligases MAFbx and MURF1 would play a significant role in
this process. Therefore, the present study analyzed the protein expression of Foxo1 and 3a, along
with their various phosphorylated forms, in addition to Foxo4, MyoG, MAFbx, and MURF1
over the torpor-arousal cycle in the cardiac muscle of thirteen-lined ground squirrels.
86
Materials and methods
Animals
All animals were captured, treated and their tissues were harvested following the same
protocol as previously described in Chapter 2. The cardiac tissue used was a mixture of atrial and
ventricular tissue.
Total Protein Extract Preparations
Samples of frozen cardiac muscle (approximately 500 mg) were homogenized for total
protein extraction as previously described. Total protein extracts were used for western blotting
experiments as previously described in Chapter 2.
Western Blotting
Western blotting was performed as described in Chapters 2 and 4 for the following six
time points: EC, EN, ET, LT, EA, IA for Foxo1, Foxo3a, their phosphorylated forms, as well as
Foxo4, MyoG, MAFbx, and MURF1. Sample aliquots containing 25 μg of protein were loaded
onto 8% [FOXO1, 3, 4, p-FOXO1 (T24), p-FOXO3 (T32), p-FOXO1 (S319), p-FOXO3a
(S318/321), MURF1] or 10% (MyoG, MAFbx) polyacrylamide gels and were run at 180 V for
60-120 min. Proteins were then wet transferred to PVDF membranes by electroblotting at 160
mA for 1.5 h. Membranes were blocked for 30 min with 5% (for Foxo1, 3a, p-Foxo1, p-Foxo3a)
or 7.5% (Foxo4, MyoG, MAFbx, MURF1) w:v milk in 1x TBST. After washing, membranes
were probed with specific primary antibodies at 4°C overnight.
87
The MyoG, MAFbx, MURF1, Foxo4 primary antibodies were the same as those used in
Chapter 5. Other primary antibodies used were: rabbit polyclonal FOXO1 (gtx110724), FOXO3a
(gtx100277), p-FOXO1/FKHR Ser319 (Genescript A00373), p-FOXO3a Ser318/321 (cs 9465), and
p-FOXO1 Thr24/p-FOXO3a Thr32 (cs 9464P). All antibodies were used at a 1:1000 v:v dilution
in 1x TBST. All blots were then incubated with HRP-linked anti-rabbit IgG secondary antibody
(Bioshop: 1:6000 v:v dilution) for 30 min at room temperature. The antibodies cross-reacted with
a single band on immunoblots at the expected molecular mass for the respective proteins as
indicated on the antibody specification sheets. Other information as in Chapter 2.
Quantification and Statistics
Band densities for Foxo1, 3a, 4, p-Foxo1, p-Foxo3a, MyoG, MAFbx, and MURF1 were
visualized and quantified as described in Chapter 2. Data are expressed as means ± SEM, n=4
independent samples from different animals.
Results
Analysis of Foxo1 and p-Foxo1 protein levels
Total Foxo1 levels immediately rose by 2.3-fold (in comparison with EC, p<0.05) upon
entering torpor (EN) and declined during torpor (ET, LT). However, Foxo1 levels were still
elevated (1.57- and 1.33-fold relative to EC, for ET and LT respectively) before decreasing
during arousal (Figure 6.1). In contrast, protein levels for phosphorylated Foxo1 at Thr24 (p-
Foxo1 T24) , one of the inhibitory phosphorylation sites targeted by Akt (Dobson et al., 2011),
declined and remained low throughout the torpor-arousal cycle (62.6%. 45%, 62%, and 77%
88
relative to EC, p<0.05 for EN, ET, LT, and EA respectively) and returned to baseline levels
during IA (Figure 6.1). Ser319 (S319) is another inhibitory phosphorylation site on Foxo1 that is
targeted by Akt (Dobson et al., 2011), p-Foxo1 S319 levels dropped upon entry and during torpor
(EN, ET, LT) by 47-54% in comparison with EC (p<0.05). Part of the Foxo1 and p-Foxo1 S319
data was contributed by Oscar Aguilar, a former student in a lab who began to study these targets
in ground squirrel but did not finish or write up this work.
Analysis of Foxo3a and p-Foxo3a protein levels
Total Foxo3a levels increased dramatically at EN and remained elevated throughout the
torpor-arousal cycle, with the peak occurring at LT (4.46-fold in comparison with EC, p<0.05)
(Figure 6.2). Akt is known to inhibit Foxo3a from translocating to the nucleus and regulating the
transcription of genes by phosphorylating Foxo3a at T32 (Dobson et al., 2011). p-Foxo3a T32
protein levels decreased dramatically by 92% relative to EC (p<0.05) at EN, and although
protein levels rose throughout torpor and during EA, the levels were still significantly decreased
in comparison with EC (74%, 66%, 41% for ET, LT, and EA respectively, p<0.05) (Figure 6.2).
p-Foxo3a S318,321 targets phosphorylation sites that are unrelated to the phosphorylation sites
targeted by common Foxo3a inhibitory proteins like Akt and JNK (Fig. 1) (Dobson et al., 2011).
p-Foxo3a S318, 321 protein levels remained steady throughout most of the torpor-arousal cycle,
but it increased dramatically during ET (2.39-fold increase relative to EC, p<0.05), contrasting
the trend observed with the other p-Foxo proteins. Part of the total Foxo3a data was contributed
by Oscar Aguilar.
Analysis of p-Foxo vs. total Foxo ratios
89
Aside from analyzing protein levels of Foxo1, 3a, and their phosphorylated forms relative
to the Coomassie blue total protein loading control, phosphorylation ratios were also obtained by
calculating and plotting the ratio of p-Foxo to total Foxo protein levels. Visualizing the data in
this manner takes into account changes in total protein levels relative to changes in
phosphorylated protein level. The p-Foxo1 S319/Foxo1 ratio showed a significant decline upon
entering and throughout torpor (80%, 69%, 63% relative to EC for EN, ET, LT respectively,
p<0.05) (Figure 6.3). The p-Foxo1 T24/Foxo1 and the p-Foxo3a T32/Foxo3a showed similar
patterns for their respective phosphorylation ratios, with dramatic declines in phosphorylation
upon entering torpor, which was sustained throughout the torpor arousal cycle (Figure 6.3). The
phosphorylation ratios decreased by 86% during EN for p-Foxo1/Foxo1 and by 97% for p-
Foxo3a/Foxo3a with respect to EC (p<0.05). The phosphorylation ratio for p-Foxo3a S318/321
declined during EN as well (76% relative to EC, p<0.05), but then the ratio increased
immediately afterwards during ET (2.85-fold relative to EN, p<0.05) (Figure 6.3).
Analysis of Foxo4 and MyoG protein levels
Foxo4 protein levels remained constant throughout the torpor-arousal cycle with the
exception of LT, where Foxo4 levels peaked and were significantly elevated in comparison with
EN (2.73-fold in comparison with EN, p<0.05) (Figure 6.4). MyoG protein levels exhibited the
same pattern of expression as Foxo4, where MyoG levels remained fairly constant throughout
the torpor-arousal cycle except for LT, where MyoG levels spiked (2.44-fold relative to EC,
p<0.05) (Figure 6.4).
Analysis of MAFbx and MURF1 protein levels
90
MAFbx protein levels remained fairly stable during EN and ET, and then suddenly
spiked during LT (3.2-fold increase in comparison with EC, p<0.05). Afterwards, MAFbx levels
remained fairly high during EA and IA (1.98- and 2.45-fold increases relative to EC for EA and
IA respectively, p<0.05) (Figure 6.5). MURF1 protein levels also remained fairly constant during
EN and ET, then it began increasing during LT and peaked during EA (1.5- and 1.8-fold
increases in comparison with EC, p<0.05) (Figure 6.5).
Discussion
The present chapter aimed at furthering our understanding of the molecular mechanisms
underlying muscle remodeling in cardiac muscle during hibernation in the 13-lined ground
squirrel (13LGS). The ground squirrel is an excellent model for studying the process of cardiac
hypertrophy as the animal enlarges its heart when its Tb falls to 4°C because increased
contractility is needed to pump colder, and more viscous blood throughout the body (Frerichs &
Hallenbeck, 1998; Frerichs et al., 1994; Nelson & Rourke, 2013). However, when bouts of the
torpor-arousal cycle end and squirrels come out of hibernation, the heart return to its regular size;
hence the process of cardiac hypertrophy is reversed following hibernation (Nelson & Rourke,
2013). Therefore, the present study focused on elucidating the mechanism behind the reversible
cardiac hypertrophy that occurs in ground squirrels. With regards to reversing cardiac
hypertrophy, we hypothesized that the family of TFs known as Foxos play a significant role in
this process. Foxos control the ubiquitin/proteasome system (UPS), which is responsible for
protein degradation, through its regulation of the E3 ubiquitin ligases MAFbx and MURF1
(Figure 1.4) (Sandri et al., 2004; Stitt et al., 2004).
91
With regard to the UPS, our data demonstrates that there is an activation of this system
during LT and arousal, leading to protein degradation and muscle atrophy. MAFbx and MURF1
both showed significant increases during LT and EA by as much as 3.2-fold from EC (p<0.05)
(Figure 6.5). These results support the hypothesis that protein degradation and atrophy of the
heart occurs naturally during arousal because squirrels are recovering from the physiological and
environmental stresses that caused enlargement of their hearts (Nelson & Rourke, 2013; Yan et
al., 2015).
In addition, under conditions of environmental stress such as glucose deprivation and
oxidative damage similar to those experienced by the 13LGS during hibernation, the expression
of ubiquitin ligases are increased through Foxo signaling (Paula-Gomes et al., 2013). For
example, TNFα-mediated activation of Foxo4 occurs as a result of ROS production. In I.
tridecemlineatus, Ros production increases as Tb is increasing following LT during arousal
(Brown et al., 2012). Therefore, our data demonstrates that there are significant upregulations in
two regulators of the E3 ligases, Foxo4 and MyoG (Moresi et al., 2010; Moylan et al., 2008;
Waddell et al., 2008). Protein expression of these two TFs showed the same pattern of significant
upregulation during LT, where Foxo4 increased 2.73-fold (p<0.05) and MyoG increased 2.44-
fold relative to EC (p<0.05) (Figure 6.4). This data also supports the idea that upregulation of
these positive regulators of MAFbx and MURF1 during LT initiates the rise in MAFbx and
MURF1 protein levels during LT and arousal – leading to atrophy and protein degradation in the
heart.
It should be noted that Foxo1 and 3a showed increased expression and activity earlier
during the torpor-arousal cycle than did Foxo4 and MyoG. Foxo1 protein levels peaked at EN
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(2.3-fold relative to EC, p<0.05) (Figure 6.1). Foxo3a on the other hand showed very high levels
throughout the torpor-arousal cycle with the exception of EC (Figure 6.2). Furthermore, the
Foxo1 amino acid residues Ser319 and Thr24 are sites that Akt/PKB can phosphorylate in order to
prevent the nuclear translocation of Foxo1; thus preventing its regulation of downstream targets.
Thr32 is an Akt phosphorylation site on Foxo3a that plays the identical role (Dobson et al., 2011).
Phosphorylation ratios of p-Foxo1 Thr24 and Ser319 both increased by at least a 63% decreases
from EC (p<0.05) during torpor. For Foxo3a, the phosphorylation ratios of Thr32 – an inhibitory
Foxo3a site, and Ser318/321 – a non-inhibitory phosphorylation site, were analyzed as well. Our
results show that p-Foxo3a Thr32 ratios decreased by over 78% from EC (p<0.05) throughout the
torpor arousal cycle, whereas decreases in the ratio of Ser318/321 increased during torpor (2.85-
fold at ET relative to EN, p<0.05) (Figure 6.3). Therefore, we conclude that there was not only
an increase in Foxo1 and Foxo3a levels during torpor, but there is also a large elevation in Foxo1
and 3a activity, as defined by an increase in the amount of dephosphorylated or nuclear Foxo.
Since p-Foxo3a Ser319 was not an inhibitory phosphorylation site targeted by Akt, it follows that
phosphorylation ratios would show less pronounced decreases than those for phosphorylation
sites targeted by Akt and a different pattern of expression during the torpor-arousal cycle.
Therefore, in the context of reversing cardiac hypertrophy during hibernation, it is likely
that Foxo4 and MyoG are important factors that regulate this process. They are the main
activators of the UPS during hibernation through upregulation of the ligases, MAFbx and
MURF1, throughout arousal. Foxo1 and 3a are other factors believed to regulate the expression
of MAFbx and MURF1 but they have also been implicated in a vast number of cellular processes
in addition to protein degradation; these processes include cell cycle inhibition, anti-oxidative
response, and a shift in cellular metabolism away from anabolic processes towards catabolic
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metabolism (Eijkelenboom & Burgering, 2013; Tessier & Storey, 2016; van der Horst &
Burgering, 2007; Wu & Storey, 2014). These processes have been shown to be activated during
hibernation in 13LGS (Wu & Storey, 2012, 2014). Therefore, Foxo1 and 3a could be expressed
earlier during the torpor-arousal cycle due to its important role in regulating functions other than
atrophy. This hypothesis could also explain the high levels of Foxo1 and 3a expression and
activity during IA despite a decline in MURF1 expression (77% in comparison to EA, p<0.05).
During arousal, there is a dramatic rise in reactive oxygen species (ROS) production associated
with oxidative thermogenesis to rewarm the body following torpor (Osborne & Hashimoto,
2006). In order to resist oxidative damage, Foxo1 and 3a must increase or maintain high levels of
expression and activity late during arousal.
In conclusion, the present study provides insight into some of the important proteins
involved in cardiac muscle remodeling during hibernation. The results from this study indicate
that there is an upregulation of the E3 ligases MAFbx and MURF1 in addition to their regulators,
mainly Foxo4 and MyoG in a coordinated fashion during late torpor and throughout arousal. The
coordination of Foxo4, MyoG, MAFbx, and MURF1 expression suggests that Foxo4 and MyoG
may be regulators that are specific to the reversal of cardiac hypertrophy, whereas Foxo1 and 3a
primarily regulate other processes such as anti-oxidant response in the heart. Therefore, in our
animals, the increase in expression of the ubiquitination machinery may occur during torpor,
albeit late, in response to an increase in protein synthesis from the hypertrophic response being
activated in the heart during early torpor (Tessier & Storey, 2012; Zhang & Storey, 2015). The
results provide support for the idea that cardiac hypertrophy causes activation of the UPS to
reduce cardiac mass and dimensions, thus reversing cardiac hypertrophy. The molecular basis of
cardiac hypertrophy occurring naturally in the hearts of 13LGS could mimic the conditions of
94
maladaptive cardiac hypertrophy and heart failure that occurs in a clinical setting, and much can
be learned from how this hibernator model reverses cardiac hypertrophy so efficiently and
naturally (Nelson & Rourke, 2013).
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Figures
Figure 6.1: Changes in the protein levels of the Foxo1 TF and its phosphorylated forms Ser319
(S319) and Thr24 (T24) over the course of the torpor-arousal cycle in cardiac muscle of I.
tridecemlineatus. Foxo1, p-Foxo1 S319, and T24 protein expression levels were visualized at six
sampling points: EC, EN, ET, LT, EA, and IA. See Chapter 2 as well as Figure 3.2 for more
information. Part of the Foxo1 and p-Foxo1 S319 timecourse data was contributed by Oscar
Aguilar, although the bands shown were from western blots performed by me.
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Figure 6.2: Changes in protein levels of the Foxo3a TF and its phosphorylated forms Ser318/321
(S318/321) and Thr32 (T32) over the course of the torpor-arousal cycle in the cardiac muscle of I.
tridecemlineatus. Other information as in Figure 3.2. Part of the Foxo3a timecourse data was
contributed by Oscar Aguilar, although the bands shown were from western blots performed by
me.
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Figure 6.3: Changes in phosphorylation ratios for phosphorylated Foxo proteins were analyzed
by taking a ratio of band densitometries between p-Foxo protein levels and total Foxo protein
levels. Phosphorylation ratios were determined for p-Foxo1 S319, T24, p-Foxo3a S318/321, and
T32. Other information as in Figure 3.2.
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Figure 6.4: Changes in protein levels of the TFs Foxo4 and MyoG over the course of the torpor-
arousal cycle in the cardiac muscle of I. tridecemlineatus. Other information as in Figure 3.2.
99
Figure 6.5: Changes in protein levels of the ubiquitin ligases MAFbx and MURF1 over the
course of the torpor-arousal cycle in the cardiac muscle of I. tridecemlineatus. Other information
as in Figure 3.2.
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Physiological adaptation to environmental changes is vital to the survival of many if not
all organisms. As a result, organisms facing extreme environmental challenges have developed a
range of adaptations to ensure their survival. One such adaptation used by some mammals in
order to survive prolonged seasonal exposure to stressful environmental conditions (i.e. lack of
food, frigid temperatures) is hibernation. The thirteen-lined ground squirrel (I. tridecemlineatus)
is an excellent example of a hibernating mammal, and by studying the molecular mechanisms
behind how it adapts to stress, we can learn a lot about how it copes with conditions (i.e. Tb at
4˚C during torpor) that are damaging if not lethal for nonhibernating mammals like humans. In
particular, the squirrel’s ability to undergo skeletal and cardiac muscle remodeling to avoid
significant losses in skeletal muscle mass and to avoid maladaptive cardiac hypertrophy are
especially unique. In fact, many studies have shown the importance and relevance of studying
the ground squirrel in as a natural model for the avoidance of skeletal muscle wasting and
cardiac hypertrophy (Cotton & Harlow, 2015; Gao et al., 2012; Li et al., 2013; Nelson & Rourke,
2013; Xu et al., 2013). Specifically, recent findings indicated that PGC-1α and the process of
mitochondrial biogenesis, which is usually active during exercise, is active as well during
hibernation and may be contributing to the preservation of muscle mass and fiber type (Xu et al.,
2013). Therefore, by improving our understanding of how ground squirrels adjust their cellular
processes, specifically transcriptional regulation, in response to changes in Tb and nutritional
stresses during hibernation, we gain an increased understanding of the molecular mechanisms of
natural muscle remodeling.
The work presented in this thesis highlights three themes in hibernation biochemistry and
molecular biology: 1) adaptations of transcription factor regulation, and 2) muscle remodeling
regulation through alterations of structural and ubiquitin ligase proteins in skeletal and cardiac
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muscle, as well as 3) the impact of environmental factors like temperature and Ca2+ on
transcription factor binding affinity. During torpor, squirrels conserve energy in all tissues by
reducing the majority of metabolic functions. This metabolic rate depression allows squirrels to
save up to 88% of active ATP expenditure, and transcription as well as translation are processes
that are suppressed to save energy (Wang & Lee, 2011). However, as demonstrated in this thesis,
the expression of certain genes need to be maintained if not elevated during torpor in order to
reduce cellular and tissue damage. Typically, unloading of skeletal muscle results in the
activation of catabolic, protein degradation networks like the ubiquitin proteasome system (UPS)
and inhibition of anabolic pathways like the insulin-mTOR pathway (Bassel-Duby & Olson,
2006; Choi et al., 2009; Glass, 2010; Malatesta et al., 2009; Rourke et al., 2004). Conversely,
cardiac muscle hypertrophy is characterized by enhanced protein synthesis and protein
degradation; resulting in the protein turnover necessary for cardiac remodeling to occur. Both of
these mechanisms are activated by increased cardiac workload due to pressure or volume (Depre
et al., 2006). These changes that occur due to the unloading of skeletal muscle and the loading of
cardiac muscle may be beneficial to meet the bodily demands of nonhibernating mammals at
first, however these mammals (i.e. humans) lack the adaptive capabilities to reverse these
processes, as a result significant skeletal muscle wasting and heart failure may occur (Bricceno et
al., 2012; Day, 2013; Frey et al., 2004; Haslett et al., 2003). This suggests that the hibernator
skeletal and cardiac muscle tissues possess unique and beneficial molecular mechanisms that
contribute to their survival and preservation.
Regulation of the NFAT-calcineurin pathway in skeletal muscle
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This thesis has identified the roles and regulation of the NFAT-calcineurin pathway and
provided evidence of the importance of this pathway to the preservation and maintenance of
skeletal muscle during hibernation. NFATc2 protein levels were elevated during torpor along
with the DNA-binding activity of NFATc1, c3, and c4. The expression pattern suggests that
NFAT TFs appear to be positively regulated during torpor as a result of the upregulation in
calcineurin and Ca2+-signaling proteins like CAM and calpain1, which activate the NFAT-
calcineurin pathway. The activation of the NFAT-calcineurin pathway during torpor led to the
positive regulation of skeletal muscle gene transcription, specifically of targets like myoferlin
that contribute to skeletal muscle growth (Demonbreun et al., 2010; Doherty et al., 2005)..
Further experiments conducted to study the effect of the cellular environment on NFAT-DNA
binding affinity showed that nuclear Ca2+ is actually an inhibitor of NFATc1-binding to DNA.
What was even more fascinating was that with decreases in temperature, like those observed
with the transition from active and arousal stages (37ºC) to torpor (4ºC) during hibernation, there
is a significant decrease in NFAT-DNA binding affinity. Furthermore, the inhibitory effect of
temperature on transcription factor binding differentially affects NFAT TFs, where NFATc3-
binding was not decreased at low temperatures to the extent that NFATc1 and c4 were.
These findings provide important insight into the molecular mechanisms that are
responsible for the preservation and/or change of muscle structure/function during hibernation,
as they are not well known aside from the finding indicating that PGC-1α is upregulated to
promote mitochondrial biogenesis, which maintains the ratio of oxidative slow-twitch muscle
fibers during torpor (Xu et al., 2013). Therefore, the present study identified that transcriptional
regulation by the NFAT TFs, which have been shown to regulate targets associated with skeletal
muscle hypertrophy and development, plays an important role in skeletal muscle remodeling
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during torpor (Armand et al., 2008; Delling et al., 2000; Hudson et al., 2014). This is evidenced
as the downstream muscle membrane protein associated with muscle development and
regeneration, myoferlin, was upregulated. In addition to myoferlin, myomaker is a membrane
protein found in the muscle that also controls myoblast fusion and results in the complete loss of
myoblast fusion when it is mutated, resulting in the absence of all skeletal muscle, which leads to
postnatal death in myomaker-null mice (Millay et al., 2013). In addition, myomaker expression
and increased myoblast fusion has been shown to occur in adult satellite cells following muscle
injury (Millay et al., 2014). The present thesis showed novel evidence that NFATc1, c3, and c4
could possibly bind to the myomaker promoter, and this novel finding could lead to further
studies that characterize NFAT regulation of this vital myogenic protein. These findings
implicate both myomaker and myoferlin as novel candidates for muscular dystrophy and Spinal
Muscular Atrophy; diseases characterized by significant muscle weakness, degeneration, and
atrophy, due to their roles in muscle formation and repair (Lorson, Rindt, & Shababi, 2010;
Marston & Hodgkinson, 2001).
Calcineurin is a calmodulin-stimulated protein phosphatase that regulates NFATs through
dephosphorylation, thereby activating and allowing NFATs to translocate to the nucleus and
regulate gene transcription (Rusnak & Mertz, 2000). CAM is a ubiquitously expressed Ca2+-
binding protein that is involved in a variety of signaling pathways that are Ca2+-dependent. It
regulates calcineurin by binding to the regulatory domain of the calcineurin A subunit when it is
exposed due to conformational changes caused by activation of the calcineurin B subunit when
there is an increase in intracellular Ca2+ levels (Klee et al., 1979; Yang & Klee, 2000). When
calcineurin B binds to Ca2+ ions, a conformational change occurs in its C-terminal autoinhibitory
domain, and the Ca2+-dependent cysteine protease calpain, specifically calpain1/calpain-µ,
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cleaves the autoinhibitory domain, thus activating calcineurin (Burkard, 2005; Lee et al., 2014;
Shioda et al., 2006). Therefore, both CAM and calpain1 are important regulators of the NFAT-
calcineurin pathway. During torpor, the amplitude of Ca2+ transients following excitation is
increased following excitation at low temperatures, and as a result, stronger contractions with
higher amplitudes are seen at lower temperatures (Liu et al., 1993; Liu et al., 1990; Wang et al.,
2000; Wang et al., 1997). Therefore, greater spikes in intracellular [Ca2+] following an action
potential and the upregulation of the above-mentioned Ca2+ proteins could be causing the
increase in NFAT activity, allowing for a maintenance of muscle mass during hibernation.
Given the extreme environmental stressors confronting 13-lined ground squirrels during
hibernation, we suspected that environmental factors such as temperature could potentially affect
the binding ability of NFATs, and potentially other TF, to DNA. Recent literature has begun to
show that gene expression could be affected by temperature, but no study has directly
investigated the temperature dependence of TF-binding to DNA (Novák et al., 2015; Chen et al.,
2015; Riehle et al., 2003; Swindell et al., 2007). Therefore, our novel finding that NFAT TFs
have lower binding affinity to target genes at lower temperatures provides evidence of another
mechanism by which hibernators undergo metabolic rate depression during torpor. Due to the
need to conserve energy, ground squirrels may enter torpor and decrease their Tb for the purpose
of decreasing transcription through an inhibition of TF binding. However, within the context of
studying the roles of different NFATs and their regulation of muscle remodeling, what is even
more fascinating is that we found differential regulation of TF binding by temperature. NFATc3
is one of the most important NFATs in terms of regulating the expression of downstream muscle
proteins like myoferlin, therefore the decrease in TF binding was reduced for this NFAT in
comparison with NFATs c1 and c4 (Delling et al., 2000; Demonbreun et al., 2010). This finding
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implies that ground squirrels downregulate gene expression of genes that play a less important
role in survival during torpor compared to genes that play more significant roles. The
thermoregulation of TFs certainly warrants further study as will be discussed in the future
directions section. However, these novel findings provide important insight into our
understanding of TFs and their regulation of gene expression, and finding how transcription
factors are thermoregulated as well as its regulation by other environmental stimuli, could lead to
tissue- and cell-specific targeting of therapeutics that activate or inhibit transcription factors.
Regulation of the NFAT-calcineurin pathway in cardiac muscle
Hibernation is also a unique and natural model whereby transitions into hibernation cause
cardiomyocyte hypertrophy to occur in a way that is comparable to the way in which human
cardiac muscle experiences hypertrophy, which results in fibrosis, hypothermia, and eventually
heart failure (Frey et al., 2004; Nelson & Rourke, 2013). This thesis has elucidated a mechanism
by which the NFAT TFs regulate cardiac hypertrophy during torpor, and the reversal of this
process during arousal. Importantly, NFATs c2 and c3 were both elevated during ET, and as a
result, myoferlin and myomaker protein levels were significantly elevated at this time point as
well. Therefore, we suspect that the end of the decline in Tb to 4˚C during torpor, which occurs at
ET, may be an important initiator for cardiac hypertrophy to occur in the hearts of these animals.
We also suspect that this upregulation of NFATc2 and c3, along with the initiation of cardiac
hypertrophy, may be regulated in part by Ca2+ signaling through the progressive increase in
calcineurin protein levels during torpor and the rise in CAM levels at EN.
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Ca2+ signaling, involving the two above-mentioned proteins in addition to calpain, appear
to play an even more significant role in the reversal of cardiac hypertrophy as the squirrel
becomes aroused. The expression of NFATc3, myoferlin, and myomaker declines following the
sharp increase at ET, and NFATc2 levels continue to progressively decline following ET and
into arousal. The decline in the expression of myoferlin and myomaker appear to be regulated by
the Ca2+ signaling proteins, especially CAM and calpain-1, whose protein levels both decline
significantly throughout the torpor-arousal cycle despite the stable levels of calcineurin. As
mentioned previously, CAM and calpain both activate calcineurin and the NFAT-calcineurin
pathway through independent mechanisms (Burkard, 2005; Klee et al., 1979; Lee et al., 2014;
Shioda et al., 2006; Yang & Klee, 2000). Therefore, decreased levels of these activators
following torpor could be causing the decline in NFATc2, c3, myoferlin, and myomaker protein
levels, thus resulting in attenuation if not reversal of cardiac hypertrophy. This pattern of
expression for CAM and calpain in cardiac muscle is in sharp contrast to what was observed in
the skeletal muscle, where these proteins were highly expressed during torpor. This is one
example of the specificity by which gene expression is controlled during hibernation in a tissue-
specific manner.
Furthermore, myomaker, a protein vital to muscle development and regeneration from
injury, was upregulated during torpor in cardiac muscle but not skeletal muscle (Millay et al.,
2013). The regulation of myomaker may depend on the severity of injury induced on the muscle,
since it was up-regulated following cardiotoxin-induced injury. Cardiotoxin is a myotoxic agent
that leads to the myolysis of the myofiber by inducing rapid plasma membrane depolarization
and the characteristics of muscle regeneration depend on the type of injury induced (Czerwinska,
Streminska, Ciemerych, & Grabowska, 2012). Therefore, the levels of skeletal muscle atrophy
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that the ground squirrel goes through during hibernation may not be enough to induce an
increase in myomaker. However, the stress induced upon the cardiac muscle during hibernation
did present a great enough stress to induce an increase in myomaker expression. In order for
cardiomyocytes to experience a reduction in size, significant atrophy and protein degradation
needs to occur, therefore we suspect that the UPS is activated as well as the squirrels are being
aroused from torpor, and these findings will be discussed in a subsequent section (Depre et al.,
2006; Galasso et al., 2010; Herrmann et al., 2007).
In summary, our findings on the importance of Ca2+-signaling in mediating activation of
the NFAT-calcineurin pathway during torpor to increase the expression of important targets like
myoferlin and myomaker that promote cardiac hypertrophy during torpor furthers our knowledge
of cardiac muscle remodeling. Furthermore, the finding that decreased expression of CAM and
calpain initiated an inhibition of NFATc2 and c3 as well as decreased expression of myoferlin
and myomaker implicates these targets in therapeutic intervention with the hopes of reversing
cardiac hypertrophy and preventing heart failure. The testing and development of inhibitors for
the Calcineurin-NFAT pathway is needed to observe for a reversal in the hypertrophic response.
The unique and tissue-specific mechanisms in ground squirrel of preserving skeletal muscle and
undergoing reversible cardiac hypertrophy during the torpor-arousal cycle make studying this
animal biologically and clinically-relevant.
Adaptations of muscle atrophy regulation in skeletal muscle
During hibernation, one would expect that muscle atrophy would occur due to
mechanical unloading, where muscles are activated less frequently and for shorter periods
especially during torpor than when squirrels are active. This would result in continual
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deterioration of skeletal muscle as this mechanical unloading stimulates protein degradation
pathways like the UPS (Herrmann et al., 2007; Schiaffino et al., 2013). However, previous work
has suggested that the ground squirrel is able to avoid significant muscle wasting during
hibernation despite the prolonged periods of mechanical unloading, especially during torpor
(Cotton & Harlow, 2015; Gao et al., 2012; Xu et al., 2013). However, there have been no studies
that have been conducted to investigate how the UPS and other mechanisms of protein
degradation are regulated during hibernation. Findings from previous studies as well as this
thesis have shown that regulators of skeletal muscle hypertrophy and remodeling, like the
NFAT-calcineurin pathway and PGC-1α regulation of mitochondrial biogenesis in muscle, are
activated during torpor. However, there is a current gap in knowledge regarding whether
pathways regulating muscle atrophy are activated during torpor as a result of inactivity, but
hypertrophic pathways like the NFAT-calcineurin pathway simply balance atrophy in order to
preserve muscle, or if muscle atrophy is downregulated during torpor, thus contributing to the
avoidance of muscle wasting.
The UPS mechanism for protein degradation occurs via ubiquitin ligases, like MAFbx
and MURF1, which ligate substrates to ubiquitin in order to target these substrates for
degradation. MyoG and Foxo4 are two important regulators of the UPS through their
transcriptional regulation of both MAFbx and MURF1 (Moresi et al., 2010; Sandri et al., 2004;
Stitt et al., 2004). Our findings indicated that Foxo4 protein levels were significantly upregulated
during torpor, whereas MyoG levels were decreased. Furthermore, similarly to MyoG, MAFbx
and MURF1 expression were decreased as well during torpor. Initially, this finding could be
interpreted as evidence that there is differential regulation of MAFbx and MURF1 by Foxo4 and
MyoG, and it is surprising that the Foxo4 expression pattern contradict those of MAFbx and
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MURF1. However, Foxo4 activation (defined by its ability to regulate transcription) as well as
its nuclear localization were studied by analyzing the levels of specific phosphorylated residues
of Foxo4. Akt/PKB inhibits the nuclear translocation and activation of Foxo4 by phosphorylating
the Ser-197 residue on Foxo4 (Matsuzaki et al., 2005; Takaishi et al., 1999). Our results indicate
the p-Foxo4 S197 levels decreased significantly throughout the torpor, suggesting that there is an
increase in Foxo4 nuclear translocation. Alternatively, the Ras-Ral pathway regulates Foxo4
transcriptional activity through the phosphorylation of Foxo4 at Thr-451 by the downstream
kinase, JNK1 (De Ruiter et al., 2001; Kops et al., 1999; Van Den Berg et al., 2013). This
mechanism is independently from the control of nuclear-cytoplasmic distribution that Akt holds
over Foxo4. p-Foxo4 T451 levels as well as the ratios of p-Foxo4/total Foxo4 both decreased
significantly during torpor, suggesting that although Foxo4 is upregulated and is able to
translocate to the nucleus, it is not transcriptionally active. Furthermore, this inactivation of
Foxo4 was shown to be regulated by the Ras-Ral pathway involving the Ras, RalA, and Ralbp1
proteins that are all downregulated during torpor.
These findings suggest that in addition to upregulating pathways that moderate and
promote hypertrophy and fiber type switching towards slow, oxidative fibers, I. tridecemlineatus
also downregulates pathways that result in protein degradation and muscle atrophy. This later
process is initiated by inhibition of the Ras-Ral pathway, as shown by decreased Ras, RalA, and
Ralbp1 protein levels during torpor, thus leading to inactivation of Foxo4 during torpor. This,
along with downregulation of MyoG, results in the decreased expression of MAFbx and MURF1
to decrease protein degradation through the UPS. These novel findings on this unique and natural
physiological process in ground squirrels advances our knowledge of skeletal muscle remodeling, and
they could be applied for therapeutic intervention in treating muscle wasting diseases like Spinal
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Muscular Atrophy and Duchenne Muscular Dystrophy. Specifically, our findings suggest that in addition
to testing agents that promote muscle growth, like Naloxone – an NFAT activator, there needs to be an
emphasis on testing inhibitors of muscle atrophy signaling pathways. For example, farnesyltransferase
inhibitors designed to block the Ras-Ral pathway for anticancer treatment could also be tested for the
possibility of inhibiting Foxo4 and reducing muscle atrophy (Yeh & Der, 2007).
Adaptations of muscle atrophy regulation in cardiac muscle
As demonstrated previously in this thesis, upregulation of the NFAT-calcineurin pathway
has been implicated in the increased synthesis of numerous proteins during cardiac hypertrophy.
With regard to protein degradation, the UPS is suspected to be an important mechanism whereby
substrates are ligated to ubiquitin via ubiquitin ligases and are targeted for degradation
(Herrmann et al., 2007). The specificity of the UPS is determined by E3 ubiquitin ligases, such
as MAFbx and MURF1, which recognize specific target proteins. The role of the UPS remains
relatively unclear in cardiac muscle in comparison with skeletal muscle; although they have
shown upregulation in association with cardiac hypertrophy or heart failure (Depre et al., 2006;
Galasso et al., 2010). This increased in expression of ubiquitination machinery may be in
response to the increase in over protein production that accompanies hypertrophy in the heart or
in response to modified or damaged proteins that need to be degraded (Day, 2013). Therefore,
with respect to reversible cardiac hypertrophy, we suspect that the UPS will be mostly active
during late torpor or arousal, especially when coming out of hibernation as perfusion and Tb will
increase; thus reversing the hypertrophic stimulus; promoting atrophy instead.
The Foxo TFs; Foxo1, 3a, 4, as well as MyoG all regulate the expression of MAFbx and
MURF1 (Moresi et al., 2010; Sandri et al., 2004; Stitt et al., 2004). Furthermore, activation of
these Foxo TFs rely upon Akt/PKB as it has been shown to block the function of all three Foxo
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proteins through phosphorylation, leading to their containment in the cytoplasm (Brunet et al.,
1999; Takaishi et al., 1999; Tang et al., 1999). Akt has numerous phosphorylation sites on
Foxo1, 3a, and 4, including Threonine32 (Thr32) for Foxo3a, as well as Thr24 and Serine319
(Ser319) for Foxo1 (Dobson et al., 2011). Our findings confirmed our hypothesis, as we found
that there is upregulation of Foxo4 and MyoG during LT. MAFbx and MURF1 followed the
same pattern of expression, where they increased in late torpor as well as arousal, thus
suggesting that the UPS is activated as ground squirrel are aroused from torpor, which could be
causing muscle atrophy and the reversal of cardiac hypertrophy.
On the other hand, we also identified increases Foxo1 and 3a protein levels as well as
decreases in inactive, phosphorylated Foxo1 and 3a proteins during torpor in comparison with
euthermic control, suggesting these Foxos are actually significantly activated during torpor.
Foxo1 and 3a are other factors believed to regulate the expression of MAFbx and MURF1 but
they have also been implicated in a vast number of cellular processes in addition to protein
degradation; these processes include cell cycle inhibition, anti-oxidative response, and a shift in
cellular metabolism away from anabolic processes towards catabolic metabolism (Eijkelenboom
& Burgering, 2013; Tessier & Storey, 2016; van der Horst & Burgering, 2007; Wu & Storey,
2014). Therefore, the present results demonstrate that the signaling pathway involving Foxo4,
MyoG, and the E3 ligases MAFbx and MURF1 play a significant role in cardiac muscle
remodeling in squirrels, which provide the molecular basis underlying the reversal of cardiac
hypertrophy, a process unique to hibernators.
Future Directions
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This thesis is the first to identify changes in NFAT-DNA binding affinity that are
temperature-dependent, although further studies need to be conducted to determine whether our
findings are specific for NFAT TFs or if it reflects a greater number of TFs. More importantly,
further studies need to determine whether the temperature-sensitivity of NFAT transcription
factors are due to conformational changes that occur at lower temperatures to the protein itself, to
DNA, or if it has to do with interactions with temperature-sensitive cofactors. For example,
NFATc2 has been shown to cooperate with heat shock transcription factor 1 (HSF1), which is
responsible for regulating the gene expression of other heat shock proteins (Hayashida et al.,
2010). Recent literature has begun to show that gene expression could be affected by
temperature, but no study has directly investigated the temperature dependence of transcription
factor-binding to DNA (Novák et al., 2015; Chen et al., 2015; Riehle et al., 2003; Swindell et al.,
2007). Most of these studies use DNA microarrays to study the global changes in gene
expression when temperature stress is induced on an organism (Riehle et al., 2003; Swindell et
al., 2007). However, although this approach identifies targets that may be involved in stress-
response, it does not directly elucidate mechanisms such as transcription factor binding affinity.
In addition to the ground squirrel’s ability to thermoregulate during torpor-arousal cycles,
they also show enhanced capabilities to maintaining intracellular Ca2+ and urea concentrations in
comparison with non-hibernating animals under the same temperature stress (Chilian &
Tollefson, 1976; Kristofferson, 1963; Liu et al., 1991; Wang & Zhou, 1999; Wang et al., 1999;
Wang et al., 2002). It was observed in this thesis that progressively increasing [Ca2+] decreased
the binding of NFATc1, whereas NFATc4 showed moderately increased binding to DNA when
[Ca2+] was increased to 600nM. These effects of intranuclear Ca2+ on NFAT-DNA binding
during torpor seem to be specific for each NFAT transcription factor, therefore this effect is
114
likely not due to the binding and blocking of DNA by intranuclear Ca2+ (Dobi & Agoston, 1998).
This effect is most likely due to Ca2+ regulation of specific export kinases like CAMKIV or
through specific coactivators of individual NFATs, such as CBP (Chawla et al., 1998; Yang et
al., 2001). However, more research needs to be conducted to further elucidate the mechanism
behind how nuclear Ca2+ regulates TF-binding to DNA.
Myoferlin and myomaker are proteins that are well-studied with regard to their
importance and contribution towards myoblast fusion, muscle hypertrophy, regeneration, and
repair (Demonbreun et al., 2010; Doherty et al., 2005; Millay et al., 2013, 2014). However, their
function in cardiac muscle is more of a mystery. Myoferlin was known to be highly expressed in
skeletal muscle and to a lesser degree in cardiac muscle, and we confirmed that it is expressed in
cardiac muscle in a pattern that is correlated with cardiac hypertrophy (Davis et al., 2000; Zhang
& Storey, 2015). Myomaker on the other hand, has only recently been discovered and its
expression has not been identified in any tissue type other than skeletal muscle. Therefore, the
current study was the first to identify the expression of myomaker in cardiac muscle. However,
its role as well as the role of myoferlin in cardiac hypertrophy and remodeling needs to be
studied further as it is known that cardiac hypertrophy and skeletal muscle hypertrophy occur via
very different mechanisms. In skeletal muscle, individual myoblast cells proliferate and
differentiate into fully mature muscle cells called myotubes, which form muscle fibers
(Bentzinger, Wang, & Rudnicki, 2012). However, in cardiac muscle, hypertrophy occurs via an
increase in the size of cardiomyocytes, and there is heightened organization of the sarcomere but
very little proliferation of cardiomyocytes occurs (Hill & Olson, 2008). Therefore, it is very
important to study the exact function of myoferlin and myomaker within cardiomyocytes using
knock-out models. Furthermore, myoferlin has been studied recently in relation to cancer in
115
multiple tissues, so this important structural protein as well as myomaker should be studied in
cell types other than muscle (Bernatchez, Sharma, Kodaman, & Sessa, 2009; Leung, Yu, Lin,
Tognon, & Bernatchez, 2013; Turtoi et al., 2013; Yu et al., 2011). Furthermore, Myomaker is
also known as Transmembrane protein 8c (TMEM8C), however there are two other members of
the family. Therefore TMEM8a and 8b should be further studied for their potential function or
lack thereof in the context of skeletal muscle and cardiac muscle remodeling.
Given the muscle remodeling that occurs over the course of the torpor-arousal cycles in
both skeletal and cardiac muscle, it is expected that nitrogen balance will be affected as a result
of the hypertrophy and protein synthesis occurring during torpor in both tissues and the protein
degradation that occurs in cardiac muscle during arousal. By measuring tissue nitrogen isotope
ratios, Lee et al. (2012) observed no changes in skeletal muscle but an increase in the isotope
ratios in heart during hibernation. This suggests that in the skeletal muscle, there is a balance
between protein breakdown and synthesis, which supports the idea that squirrels are able to
maintain and preserve muscle mass during hibernation. On the other hand, in the heart, the
increase in the nitrogen isotope ratio indicates that there is greater protein synthesis than
breakdown occurring in this organ (Lee et al., 2012). This also supports the idea that cardiac
hypertrophy occurs during hibernation. However, the study of nitrogen balance needs to be
extended to timecourse experiments in order to understand how nitrogen balance is affected by
the constantly-changing molecular stimuli for protein synthesis and degradation that occurs over
the torpor-arousal cycle.
In summary, this thesis studied the molecular response of striated muscles to the natural
process of hibernation, which is characterized by metabolic rate depression, cold body
116
temperatures, and drastic physiological changes. The analysis of the NFAT transcription factors,
along with their regulation through calcium signaling, and their downstream targets (myoferlin,
myomaker), has shown the importance of the NFAT-calcineurin pathway in ensuring the
survival of the organism and in the avoidance of disuse-induced muscle atrophy as well as in
regulating reversible cardiac hypertrophy. Furthermore the regulation of NFAT TFs by
environmental stimuli such as temperature and intranuclear [Ca2+] suggested that these TFs as
well as others may be regulated by both conditions. The analysis of the Foxo family of TFs, its
regulation through the Akt and Ras-Ral pathways, the MyoG TF, in addition to the ubiquitin
ligases MAFbx and MURF1, indicated that downregulation of muscle atrophy in skeletal muscle
during torpor and in cardiac muscle during arousal contribute to the avoidance of skeletal muscle
loss and in reversing cardiac hypertrophy during and after hibernation, respectively. Clearly the
process of hypometabolism in the thirteen-lined ground squirrel is a complex process that is
highly regulated. However, by comparing the effective physiological adaptations in this system
during hibernation to humans provides insight into how we can combat certain diseases and live
efficiently like squirrels.
118
Research Articles Accepted
Yichi Zhang, Shannon N. Tessier, Kenneth B. Storey. 2016. Inhibition of skeletal muscle
atrophy during torpor in ground squirrels occurs through downregulation of MyoG and
inactivation of Foxo4. Cryobiology. Accepted.
Yichi Zhang, Oscar Aguilar, Kenneth B. Storey. 2016. Transcriptional activation of muscle
atrophy promotes cardiac muscle remodeling during mammalian hibernation. PeerJ, 4,
e2317. DOI:10.7717/peerj.2317.
Yichi Zhang, Kenneth B. Storey. 2016. Regulation of gene expression by NFAT transcription
factors in hibernating ground squirrels is dependent on the cellular environment. Cell
Stress and Chaperones, 21, 883-894. DOI:10.1007/s12192-016-0713-5.
Yichi Zhang, Kenneth B. Storey. 2015. Expression of nuclear factor of activated T cells (NFAT)
and downstream muscle-specific proteins in ground squirrel skeletal and heart muscle
during hibernation. Molecular Cell Biochemistry, 412, 27-40. DOI:10.1007/s11010-015-
2605-x.
Research Articles in Review
Rasha Al-attar, Yichi Zhang, Kenneth B. Storey. Osmolyte regulation by TonEBP/NFAT5
during anoxia-recovery and dehydration-rehydration stresses in the freeze-tolerant wood
frog (Rana sylvatica). PeerJ. In Review.
Conference Papers
119
Zhang Y, Storey KB (2015) Expression of nuclear factor of activated T cells (NFAT) and
downstream muscle-specific proteins in ground squirrel cardiac muscle. Canadian Journal
of Cardiology, 31(10), S143. doi:10.1016/j.cjca.2015.07.311
Other Peer-Reviewed Publications
Zhang Y (2015) Epigenomics: biological and clinical implications of histone deacetylation.
Health Science Inquiry, 6.
Communications and Scientific Meetings
Poster Presentations
Zhang Y, Tessier SN, Storey KB. Expression of nuclear factor of activated T-cells (NFAT) and
downstream muscle-specific proteins in ground squirrel skeletal and heart muscle during
hibernation, Cryobiology 2016 Conference, Ottawa, Ontario. July, 2016.
Zhang Y, Aguilar AA, Storey KB. Transcriptional activation of muscle atrophy promotes cardiac
muscle remodelling during mammalian hibernation, Ottawa-Carleton Institute of Biology
Conference, Ottawa, Ontario. April, 2016.
Zhang Y, Aguilar AA, Storey KB. Transcriptional activation of muscle atrophy promotes cardiac
muscle remodelling during mammalian hibernation. 18th Annual Chemistry and
Biochemistry Graduate Research Conference, Montreal, Quebec. November, 2015.
Zhang Y, Storey KB. Expression of nuclear factor of activated T cells (NFAT) and downstream
muscle proteins in ground squirrel cardiac muscle. Canadian Cardiovascular Congress,
Toronto, Ontario. October, 2015.
120
Zhang Y, Storey KB. Expression of nuclear factor of activated T cells (NFAT) and downstream
targets in ground squirrel skeletal muscle during hibernation. 21st Annual Canadian
Connective Tissue Conference, Quebec City, Quebec. May, 2015.
Zhang Y, Storey KB. Expression of the Nuclear Factor of Activated T cells and downstream
targets in ground squirrel cardiac muscle, Ottawa-Carleton Institute of Biology
Conference, Ottawa, Ontario. April, 2015.
Zhang Y, Storey KB. Expression of the Nuclear Factor of Activated T cells and muscle-specific
targets in ground squirrel cardiac muscle, Ottawa Heart Research Conference, Ottawa,
Ontario. April, 2015.
Oral Presentations
Expression of nuclear factor of activated T cells (NFAT) and downstream targets in ground
squirrel skeletal muscle during hibernation, Ottawa-Carleton Institute of Biology
Conference, Ottawa, Ontario (Oral).
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Appendix A: Antibodies Used for Western Blotting
Protein Target Supplier Associated Product Code
NFATc1 Santa Cruz sc-13033
NFATc2 Santa Cruz sc-13024
NFATc3 Santa Cruz sc-8321
NFATc4 Santa Cruz sc-13036
Myomaker/TMEM8c Santa Cruz sc-244460
Myoferlin Santa Cruz sc-134798
Foxo1 Genetex GTX110724
Foxo3a Genetex GTX100277
p-Foxo1 S319 Genescript A00373
p-Foxo1 T24/p-Foxo3a T32 Cell Signaling 9464P
p-Foxo3a S318/321 Cell Signaling 9465
Foxo4 Cell Signaling 9472
p-Foxo4 S197 Santa Cruz sc-101628
p-Foxo4 T451 Signalway Antibody 12053
MAFbx Santa Cruz sc-27645
MURF1 Genetex GTX110475
MyoG Santa Cruz sc-576
Calcineurin A Genetex GTX111039
CAM Upstate Biotechnology 06-396
Calpain-1 Genetex GTX102340
Ras Genetex GTX132480
Ral A Genetex GTX114204
Ralbp1 Signalway Antibody 38202
147
Appendix B: Western Blotting Conditions
1˚
antibody
target
Tissue Protein
molecular
weight
(kDa)
Gel
percent
(%)
Transfer
conditions
and time (h)
Block
with
milk
(%)
1˚ antibody
incubation
2˚ antibody
incubation
NFATc1 Muscle 101 6 160 mA, 3h 2.5 1:500 overnight Rabbit 1:6000 30 min
Heart 101 6 160 mA, 3h 2.5 1:500 overnight Rabbit 1:6000 30 min
NFATc2 Muscle 92 6 160 mA, 3h 2.5 1:500 overnight Rabbit 1:6000 30 min
Heart 92 6 160 mA, 3h 2.5 1:500 overnight Rabbit 1:6000 30 min
NFATc3 Muscle 130 6 160 mA, 3h 2.5 1:500 overnight Rabbit 1:6000 30 min
Heart 130 6 160 mA, 3h 2.5 1:500 overnight Rabbit 1:6000 30 min
NFATc4 Muscle 85 6 160 mA, 3h 2.5 1:500 overnight Rabbit 1:6000 30 min
Heart 85 6 160 mA, 3h 2.5 1:500 overnight Rabbit 1:6000 30 min
Myomaker/
TMEM8c
Muscle 235 15 160 mA, 1.5h 5 1:500 overnight Goat 1:6000 30 min
Heart 235 15 160 mA, 1.5h 5 1:500 overnight Goat 1:6000 30 min
Myoferlin Muscle 25 6 160 mA, 3h 5 1:500 overnight Rabbit 1:6000 30 min
Heart 25 6 160 mA, 3h 5 1:500 overnight Rabbit 1:6000 30 min
Foxo1 Muscle 68 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Heart 68 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Foxo3a Muscle 90 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Heart 90 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
p-Foxo1
S319
Muscle 68 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Heart 68 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
p-Foxo1
T24/p-
Foxo3a
T32
Muscle Foxo1: 69,
Foxo3a:
95
8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Heart 69/95 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Muscle 72 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
148
p-Foxo3a
S318/321
Heart 72 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Foxo4 Muscle 53 8 160 mA, 1.5h 7.5 1:1000 overnight Rabbit 1:6000 30 min
Heart 53 8 160 mA, 1.5h 7.5 1:1000 overnight Rabbit 1:6000 30 min
p-Foxo4
S197
Muscle 53 10 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Heart 53 10 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
p-Foxo4
T451
Muscle 53 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Heart 53 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
MAFbx Muscle 42 10 160 mA, 1.5h 7.5 1:1000 overnight Rabbit 1:6000 30 min
Heart 42 10 160 mA, 1.5h 7.5 1:1000 overnight Rabbit 1:6000 30 min
MURF1 Muscle 40 8 160 mA, 1.5h 7.5 1:1000 overnight Rabbit 1:6000 30 min
Heart 40 8 160 mA, 1.5h 7.5 1:1000 overnight Rabbit 1:6000 30 min
MyoG Muscle 34 10 160 mA, 1.5h 7.5 1:1000 overnight Rabbit 1:6000 30 min
Heart 34 10 160 mA, 1.5h 7.5 1:1000 overnight Rabbit 1:6000 30 min
Calcineurin
A
Muscle 59 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Heart 59 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
CAM Muscle 19 15 30 V 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Heart 19 15 30 V 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Calpain-1 Muscle 82 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Heart 82 8 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Ras Muscle 21 15 30 V 1.5h 7.5 1:1000 overnight Rabbit 1:6000 30 min
Heart 21 15 30 V 1.5h 7.5 1:1000 overnight Rabbit 1:6000 30 min
Ral A Muscle 24 15 30 V 1.5h 7.5 1:1000 overnight Rabbit 1:6000 30 min
Heart 24 15 30 V 1.5h 7.5 1:1000 overnight Rabbit 1:6000 30 min
Ralbp1 Muscle 74 10 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
Heart 74 10 160 mA, 1.5h 5 1:1000 overnight Rabbit 1:6000 30 min
147
Appendix C: Summary Figures and Tables
Supplementary Figure 1: Summary figure of the relationship between the targets analyzed in the current
thesis. The NFAT-calcineurin pathway shown at the top of the figure regulates the downstream muscle
proteins Myoferlin and Myomaker, which may result in increased myoblast differentiation. The
transcription factors Myogenin and the Foxo family of transcription factors regulate the E3 ubiquitin
ligases MAFbx and MuRF1. These ligases facilitate the degradation of a many proteins involved in a host
of cellular processes like protein synthesis and myoblast differentiation. This suggests that the regulation
of these ligases could control the process of muscle atrophy.
148
Supplementary Table 1: Table summarizing the changes that took place with the western blotting results
of the NFAT-calcineurin pathway and their downstream muscle-specific proteins in skeletal muscle. The
torpor-arousal cycle including entry into torpor (EN), deep torpor (ET, LT), and arousal (EA, LA, IA)
were analyzed in comparison with euthermic control (EC). The green upwards arrow indicates
upregulation of protein expression, the red downwards arrow indicates downregulation of protein
expression, and the sideways arrow indicates no significant change.
149
Supplementary Table 2: Table summarizing the changes that took place with the DPI-ELISA results of
NFATc1, c3, and c4 in skeletal muscle. The torpor-arousal cycle including EN, deep torpor (ET, LT), and
arousal (EA, LA, IA) were analyzed in comparison with EC. The green upwards arrow indicates
upregulation of protein expression, the red downwards arrow indicates downregulation of protein
expression, and the sideways arrow indicates no significant change.
150
Supplementary Table 3: Table summarizing the changes that took place with the western blotting results
of the NFAT-calcineurin pathway and their downstream muscle-specific proteins in cardiac muscle. The
torpor-arousal cycle including EN, deep torpor (ET, LT), and arousal (EA, LA, IA) were analyzed in
comparison with EC. The green upwards arrow indicates upregulation of protein expression, the red
downwards arrow indicates downregulation of protein expression, and the sideways arrow indicates no
significant change.
151
Supplementary Table 4: Table summarizing the changes that took place with the western blotting results
of the Ras-Ral pathway, Foxo4 and its phosphorylated forms, as well as Myogenin their downstream E3
ubiquitin ligase proteins in skeletal muscle. The torpor-arousal cycle including EN, deep torpor (ET, LT),
and arousal (EA, LA, IA) were analyzed in comparison with EC. The green upwards arrow indicates
upregulation of protein expression, the red downwards arrow indicates downregulation of protein
expression, and the sideways arrow indicates no significant change.
152
Supplementary Table 5: Table summarizing the changes that took place with the western blotting results
of Foxo1, Foxo3a, Foxo4 and its phosphorylated forms, as well as Myogenin their downstream E3
ubiquitin ligase proteins in cardiac muscle. The torpor-arousal cycle including EN, deep torpor (ET, LT),
and arousal (EA, LA, IA) were analyzed in comparison with EC. The green upwards arrow indicates
upregulation of protein expression, the red downwards arrow indicates downregulation of protein
expression, and the sideways arrow indicates no significant change.
153
Appendix D: Summary of Contributions
I have gathered, quantified, analyzed, and interpreted all of the data in this thesis with the exception of
selected proteins analyzed via western blotting in Figures 5.3, 6.2, and 6.3. The MAFbx timecourse
western blots in Figure 5.3 were done by Dr. Shannon N. Tessier and are unpublished and not part of her
thesis. She contributed her data on this target as it was vital to the present chapter and she is a co-author
on our submitted manuscript for publication. In Figures 6.2 and 6.3, part of the Foxo3a timecourse data
was contributed by Oscar A. Aguilar, although the bands shown were from western blots performed by
me.